Christoph Dobeš

Sexual reproduction, hybridization, apomixis, and polyploidization in the genus Boechera (Brassicaceae)

Of the 340 genera in the Brassicaceae, apomictic reproduction is found only in the North American genus Boechera. We investigated phylogenetic relationships, ability to hybridize, mating system, and ploidy levels of 92 lines sampled from 85 populations and representing 19 Boechera species. Phylogenetic analyses based on chloroplast DNA sequences identified three lineages in the genus. Reciprocal crosses of each line were made to a common sexual diploid B. stricta tester. The resulting F(1) progeny were analyzed for the inheritance of polymorphic microsatellite loci, genome size, and seed production. Intraspecific B. stricta crosses confirmed that this species is mostly diploid and sexual. Interspecific crosses revealed many other species were diploid and sexual and could be successfully hybridized with the tester. We also found obligate and facultative apomictic diploid and triploid lines. De novo F(1) polyploids (either triploids or tetraploids) were derived from the union of nonreduced (from an apomictic parent) and reduced (from the tester) gametes. However, seed production of these F(1) plants was generally low, suggesting a failure in the transmission of apomixis. The creation of a wide array of segregating genetic populations will facilitate future research on the evolution and inheritance of quantitative variation in Boechera.

Phylogeography of a living fossil: Pleistocene glaciations forced Ginkgo biloba L. (Ginkgoaceae) into two refuge areas in China with limited subsequent postglacial expansion

Ginkgo biloba is one of the most famous and therefore intensively studied living fossils. The Ginkgoaceae was suggested to have originated in the early Permian approximately 280mya, and at the height of their worldwide radiation consisted of least 16 genera that were distributed in temperate forests. However, Chinas G. biloba is the only species of this genus which has survived until today. Using a phylogeographical approach we analyzed the history of this remnant from the past. G. biloba is native to China and has been distributed less than 300 years ago by humans throughout all temperate regions of the world. Separate from this human influence, native Chinese populations of G. biloba have been affected by the cycles of glaciation and deglaciation of the Pleistocene era, similar to many other global plant species. We used molecular markers from the nuclear (AFLPs) and plastid genomes (trnK intron, trnS-trnG intergenic spacer), and based on these data, glacial survival of Gingko populations within two refugia in Southwestern and Eastern China, respectively, is suggested. Specifically, for the first time, this study provides evidence of the existence of a refuge area in Eastern China on the West Tianmu Mountains. We can not support any extensive postglacial range expansion from these refuge areas. Furthermore, AFLP data indicate substantial genetic differentiation among populations from Southwestern, Eastern and Central China. We also hypothesize that this evidence supports that as a long-lived dioecious plant, G. biloba displays high levels of intra-populational genetic diversity that have been maintained in most populations, regardless if they are native or reforested stands. Furthermore, we provide evidence that planted trees from Europe, Japan, Korea and America were introduced multiple times from Eastern China.

Non-coding nuclear DNA markers in phylogenetic reconstruction.

Molecular DNA based data sets are the most important resource for phylogenetic reconstruction. Among the various marker systems, which were introduced and optimized within the last decade, coding sequences played an important role, especially when molecular clock approaches and multi-gene datasets were assembled. However, non-coding DNA sequences do not only play a quantitatively dominant role, as demonstrated by the two examples nuclear ITS (Internal transcribed spacer regions of nuclear ribosomal DNA) and plastidic trnL-F region, but there is also a wide range of different marker systems that can be applied in different ways. Herein, we review the application of several non-coding nuclear DNA marker systems for phylogenetic reconstructions and summarize valuable information for future research.

Intraspecific diversification in North American Boechera stricta (= Arabis drummondii), Boechera ×divaricarpa, and Boechera holboellii (Brassicaceae) inferred from nuclear and chloroplast molecular markers—an integrative approach

We performed a combined evolutionary analysis of North American Boechera stricta, Boechera holboellii, and their hybrid Boechera ×divaricarpa using information on ploidy level estimators, allelic microsatellite variation, noncoding regions of the plastidic genome (cpDNA), and sequences of the internal transcribed spacers 1 and 2 of the nuclear ribosomal DNA (ITS). Somatic ploidy levels of herbarium specimens were estimated based on comparison of pollen size and the number of alleles per locus at seven microsatellites. Results indicate that B. stricta and B. holboellii are genetically distinct from each other, although we also find evidence for occasional introgression between both parental species. Microsatellite patterns for B. stricta from northeastern North America are genetically distinct from western populations, suggesting isolation in glacial refugia along the southeastern margin of the continuous ice shield. Microsatellites supported recent origin of B. ×divaricarpa. Correspondence of nrDNA with cpDNA genetic variation for the majority of diploid B. holboellii accessions suggests a basal, sexual evolutionary unit within a polymorphic B. holboellii group. Hybridization of genetically distinct lineage(s) evidently played an important role in the establishment of polyploid B. holboellii. Frequency of polyploid B. holboellii is substantially higher in the southern United States. This trend corresponds to a southerly distribution of derived chloroplast haplotypes, suggesting an evolutionary advantage of polyploidy and associated apomixis in the colonization of the Sierra Nevada and the Southern Rocky Mountains.

A comprehensive chloroplast DNA-based phylogeny of the genus Potentilla (Rosaceae): implications for its geographic origin, phylogeography and generic circumscription.

A reconstruction of phylogenetic relationships based on three chloroplast DNA markers comprising 98 species of the genus Potentilla and 15 additional genera from the tribe Potentilleae (Rosaceae) is presented. The phylogeny supported the current generic concept of the subtribe Fragariinae and resolved major lineages within the subtribe Potentillinae. Resolved lineages corresponded to the combined genera Argentina, Piletophyllum, and Tylosperma, an European and Asian group of species approximately equivalent to the Trichocarpae Herbaceae sensu Th. Wolf, the series Tormentillae Th. Wolf, a North American clade comprising the genera Horkelia, Horkeliella, and Ivesia, the species Potentilla fragarioides and P. freyniana, and to a taxonomically highly diverse but molecularly little diverged core group of Potentilla, respectively. Age estimates of phylogenetic splits resolved in the Potentilleae using Bayesian inference, suggested a diversification of the tribe in the Eocene and radiation of two major evolutionary lineages corresponding to the Fragariinae and Potentillinae at approximately comparable times. Ancestral area reconstructions based on the recent distribution ranges of species and collection sites of cpDNA haplotypes suggested an Asian origin for Potentilla s.str., and explained the arrival of this still informal taxon in Europe and particularly North America by multiple dispersal events. In combining the phylogenetic and geographic data with molecularly inferred time estimates and taxonomy, strongly contrasting evolutionary patterns were identified. These evolutionary patterns included rapid speciation on a continental and worldwide scale accompanied by multiple intercontinental dispersals opposed to largely diverged lineages of limited taxonomic diversity and vicariant geographic distribution. The molecular-based phylogeographic hypothesis finally is discussed on the background of the fossil record of Potentilla.

Phylogeographic structure of the chloroplast DNA gene pool in North American Boechera - a genus and continental-wide perspective.

Continental-wide phylogeographic studies of plants in North America are rare. In our study we examined the phylogeographic history of Boechera (Brassicaceae) on a continental-wide scale testing if it is possible to do an analysis for 57 of the currently accepted taxa simultaneously. A large amount of haplotype sharing is explained both by recurrent hybridization and by non-differentiation of haplotypes since speciation. Hence, the chloroplast gene pool in Boechera predates speciation and therefore justifies the simultaneous analysis of a large number of taxa. Unrelated from taxon identity we can show that the evolutionary lineages detected have a different phylogeographic history in terms of glacial refugia and recently recolonised areas.

Isozyme and morphological diversity within apomictic and sexual taxa of the Ranunculus auricomus complex

Abstract. In 17 populations of the tetraploid agamospecies R. variabilis, 10 enzyme systems have been studied by horizontal starch gel electrophoresis, and compared to previous data of 3 populations of the diploid sexual species R. notabilis. Allozymic and genotypic diversity of 12 polymorphic loci, and multilocus genotype diversity in R. variabilis compares to other apomictic taxa, confirming the assumed apomictic mode of reproduction previously found in 3 populations. A cluster analysis based on genetic distance values separated the two taxa, the populations of southeastern Austria sympatric with R. notabilis showing no closer relationships to the sexual species than the allopatric ones. The R. variabilis populations of the circumalpine regions are separated as a rather uniform group, those of the Bohemian massif as another, more heterogeneous group. This geographic differentiation is mainly due to genotypic variation, which is higher in the Bohemian massif than in the circumalpine group, and lower in areas where R. variabilis is sympatric with R. notabilis. Proportion of multilocus genotypes within populations (G/N), genotype diversity (D), and genotype evenness (E) measures indicate facultative recombination events with fixation and clonal reproduction of new recombined genotypes, which is regarded as the most important factor for the evolution of new lineages in goldilocks. Morphometric data of the R. variabilis and R. notabilis populations studied by cluster and principal coordinate analyses clearly separated the two taxa, and indicated a slight geographic differentiation with in R. variabilis corresponding to the results of isozyme data. A principal coordinate analysis of R. variabilis individuals based on stem and fruit characters showed a clinal variation, but no clear separation of groups. The variation found in this PCoA is almost completely covered from the most widespread clone (genotype I), and also from individual genotypes. The implications of the results on different species concepts for apomicts are discussed. For R. variabilis, no taxonomic conclusions can be drawn from the present data set.

Evolution of trnF(GAA) pseudogenes in cruciferous plants

In several studies we used the 5′-trnL(UAA)–trnF(GAA) region of the chloroplast DNA for phylogeographic reconstructions, gene diversity calculations and phylogenetic analyses among the genera Arabidopsis and Boechera. Despite the fact that extensive gene duplications are rare within the chloroplast genome of higher plants, within several genera of the Brassicaceae the anticodon domain of the trnF(GAA) gene exhibit extensive gene duplications with 1–12 tandemly repeated copies in close 5′-proximity of the functional gene. A recent re-examination and additional analysis of trnL(UAA)–trnF(GAA) regions from numerous cruciferous taxa not only reveal extensive trnF gene duplications, but also favour the hypothesis that in cruciferous taxa at least four independent phylogenetic lineages are characterized by these pseudogenes. Among these lineages there is one major clade of taxa carrying pseudogenes indicating an ancient split in crucifer evolution. In two case studies, Boechera and Arabidopsis, intra- and inter-molecular recombinations have been shown to be the reason for the reciprocal exchange of several similar motifs. However, functional constraints might favour two to three or five to six copies as shown for Arabidopsis and Boechera. Herein, we compare the occurrence and distribution of pseudogene copy number in the framework of a comprehensive survey of cpDNA haplotype variation in Boechera, the former genus Cardaminopsis and Arabidopsis thaliana and comment on the value of such kind of mutations in phylogenetic and evolutionary reconstructions.

EMBRYOLOGY, KARYOLOGY, AND MODES OF REPRODUCTION IN THE NORTH AMERICAN GENUS BOECHERA (BRASSICACEAE): A COMPILATION OF SEVEN DECADES OF RESEARCH1

Abstract The North American–Greenlandic genus Boechera Á. Löve & D. Löve (Brassicaceae) is distinguished from other phylogenetic lineages recognized within the artificial taxon Arabis L. s.l., in which it has formerly been included, by its base chromosome number x = 7. Based on outgroup comparisons, we consider this chromosome number to be derived from an ancestral genome comprising eight chromosomes. Chromosome counts are now available for about half of the species names listed in the most recent taxonomic treatments of the genus. An analysis of the karyological differentiation with respect to chromosome numbers was performed, and these patterns were correlated with data on pollen viability and population genetic measures in order to deduce modes of reproduction. This approach allowed us to distinguish three main groups: sexual, amphi-apomictic, and apomictic species, respectively. We focused further on the cytology of gametes, especially on that of pollen, and we discuss its role in the formation of new genotypes and cytotypes. Evidence for the following evolutionary phenomena is currently available: (1) allopolyploidization, (2) homoploid hybrid formation, and (3) establishment of viable offspring by means of fusion of highly aneuploid gametes, which resulted from irregular meiotic divisions. In contrast, there is no strict evidence demonstrating the occurrence of autopolyploidization. Boechera experts have just started to integrate evolutionary concepts derived from molecular analyses into taxonomic research, and profound changes, with respect to species definition and circumscription, are to be expected in the near future. The identified modes of reproduction and evolutionary phenomena will contribute to the development of a theoretical and practical basis on which future taxonomic classifications and species concepts can be based.

Prospects and limits of the flow cytometric seed screen – insights from Potentilla sensu lato (Potentilleae, Rosaceae)

The flow cytometric seed screen allows for identification of reproductive modes of seed formation and inference of the ploidy of contributing gametes. However, the lack of a mathematical formalization to infer male/female genomic contributions, and the prerequisite of a binucleate female contribution to the endosperm limits its applicability. We evaluated this assumption combining a DNA-based progeny survey with a comparison of the cytology of reproductive pathways co-occurring within single individuals representing 14 Potentilleae species from six phylogenetic lineages. A numerical framework valid for sexual and pseudogamous taxa was developed, enabling quantification of female and male genomes contributing to embryo and endosperm independent of gametophyte origins, numbers of sperm involved and ploidy of parents. The inference strongly depended on accurate peak index estimation. The endosperm of Potentilleae species received a binucleate female contribution in five evolutionary lineages whereas endosperm formation remained uncertain in the Tormentillae. A modified flow cytometric seed screen protocol was developed to cope with low endosperm contents. Evolutionary conservation of a binucleate female contribution to the endosperm suggested wide applicability of flow cytometric seed screen – at least in the Potentilleae. However, alternative progeny surveys and precise embryo/endosperm ploidy estimates are required for a comprehensive understanding of the cytology of seed formation.