Barbara Neuffer

Recent speciation associated with the evolution of selfing in Capsella

The evolution from outcrossing to predominant self-fertilization represents one of the most common transitions in flowering plant evolution. This shift in mating system is almost universally associated with the “selfing syndrome,” characterized by marked reduction in flower size and a breakdown of the morphological and genetic mechanisms that prevent self-fertilization. In general, the timescale in which these transitions occur, and the evolutionary dynamics associated with the evolution of the selfing syndrome are poorly known. We investigated the origin and evolution of selfing in the annual plant Capsella rubella from its self-incompatible, outcrossing progenitor Capsella grandiflora by characterizing multilocus patterns of DNA sequence variation at nuclear genes. We estimate that the transition to selfing and subsequent geographic expansion have taken place during the past 20,000 years. This transition was probably associated with a shift from stable equilibrium toward a near-complete population bottleneck causing a major reduction in effective population size. The timing and severe founder event support the hypothesis that selfing was favored during colonization as new habitats emerged after the last glaciation and the expansion of agriculture. These results suggest that natural selection for reproductive assurance can lead to major morphological evolution and speciation on relatively short evolutionary timescales.

Recent speciation of Capsella rubella from Capsella grandiflora, associated with loss of self-incompatibility and an extreme bottleneck

Flowering plants often prevent selfing through mechanisms of self-incompatibility (S.I.). The loss of S.I. has occurred many times independently, because it provides short-term advantages in situations where pollinators or mates are rare. The genus Capsella, which is closely related to Arabidopsis, contains a pair of closely related diploid species, the self-incompatible Capsella grandiflora and the self-compatible Capsella rubella. To elucidate the transition to selfing and its relationship to speciation of C. rubella, we have made use of comparative sequence information. Our analyses indicate that C. rubella separated from C. grandiflora recently (≈30,000–50,000 years ago) and that breakdown of S.I. occurred at approximately the same time. Contrasting the nucleotide diversity patterns of the 2 species, we found that C. rubella has only 1 or 2 alleles at most loci, suggesting that it originated through an extreme population bottleneck. Our data are consistent with diploid speciation by a single, selfing individual, most likely living in Greece. The new species subsequently colonized the Mediterranean by Northern and Southern routes, at a time that also saw the spread of agriculture. The presence of phenotypic diversity within modern C. rubella suggests that this species will be an interesting model to understand divergence and adaptation, starting from very limited standing genetic variation.

Colonization history and introduction dynamics of Capsella bursa-pastoris (Brassicaceae) in North America: isozymes and quantitative traits

Multilocus isozyme genotypic composition for aspartate aminotransferase (AAT), leucine aminopeptidase (LAP) and glutamate dehydrogenase (GDH) was studied for Capsella in the source continent, Europe (9000 plants from 593 populations), and in the colonized continent, North America (2700 plants from 88 populations). North America was depauperate in the number of genotypes (by ≈ 50%), but in terms of frequencies, a few genotypes were common and shared by both continents. Although some, very rare, genotypes were, however, unique for North America, our data provided no evidence to indicate that the introduced gene pools were reconstructed on a multilocus genetic basis after introduction. Instead, they argued for a considerable number of independent introduction events. Geographical distribution patterns of multilocus genotypes in Europe and North America were pronounced and enabled us to trace the colonization history of Californian Capsella back to Spanish ancestral populations and those of temperate North America back to temperate European gene pools. A random‐block field experiment with 14 Californian populations from different climatic regions revealed that variation patterns of quantitative traits reflect ecotypic variation, and the ecological amplitude of Capsella in North America is similar to that in Europe, which can be traced back to the introduction of preadapted genotypes. It appears that certain multilocus isozyme genotypes are associated with certain ecotypes. The variable European gene pool of Capsella was essentially introduced into North America without major genetic changes.

Hybridization and evolution inCardamine (Brassicaceae) at Urnerboden, Central Switzerland: Biosystematic and molecular evidence

Hybridization between two diploid (2n = 2x = 16) species ofBrassicaceae, Cardamine rivularis andC. amara, at Urnerboden, Central Switzerland, resulted in the rather unusual triploid hybridC. insueta (2n = 3x = 24), and later on in the amphiploidC. schulzii (2n = 6x = 48). The hybrid and the neopolyploid species colonized successfully some man-made biotopes. Plants ofC. insueta are mostly functional females with non-dehiscent anthers, but true hermaphrodite individuals with partly sterile pollen grains also occur within the population. Analyses of cpDNA and nuclear DNA permitted to establish the parentage of the hybrid: the maternal parent which contributed unreduced egg cells proved to beC. rivularis whereas the normally reduced pollen originated fromC. amara. The pronounced genetic variability inC. insueta revealed by isozyme and RAPD analyses, at variance with the polarized segregation, heterogamy and strong vegetative reproduction of the hybrid, is possibly influenced by recurrent formation ofC. insueta which party results from backcrosses betweenC. insueta andC. rivularis but may also proceed by other pathways. The amphiploidCardamine schulzii has normally developed anthers but its pollen is sometimes highly sterile. The surprisingly uniform genetic make-up of the new amphiploid species might be related to its possible monotopic origin and/or young phylogenetic age but should be further assessed. Site management seems to be very important to a further development of hybridogenous populations and their parent species. In conclusion, the evolution at Urnerboden is discussed in the context of the traditional concept of multiple plant origins.

Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa

Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region for 189 accessions representing 184 species in 121 genera of Brassicaceae were used to determine monophyly of tribes and genera, tribal boundaries, and component genera. Parsimony analysis and Bayesian inference suggest that the tribes Camelineae and Arabideae are polyphyletic and should be subdivided into smaller tribes. The study also supports the recent recognition of the new tribes Aphragmeae, Biscutelleae, Buniadeae, Calepineae, Conringieae, Dontostemoneae, Erysimeae, Malcolmieae, Megacarpaeeae, and Turritideae. The data argue for the placement of Borodinia in the tribe Boechereae, Litwinowia and Pseudoclausia in the Chorisporeae, Atelanthera and Streptoloma in the Euclidieae, and Megacarpaea and Pugionium in the Megacarpaeeae, and exclusion of Asperuginoides, Didymophysa, and Ptilotrichum from the Alysseae, Macropodium, Pseudoturritis, and Stevenia from the Arabideae, and Crucihimalaya, Irenepharsus, Pachycladon, and Turritis from the Camelineae. Finally, the findings support the expansion of Stevenia to include both Berteroella and Ptilotrichum, Sterigmostemum to include Oreoloma and one species of Anchonium, Crucihimalaya to include Transberingia and several species of Arabis, and Parrya to include Pseudoclausia. The data also suggest that Calymmatium and Olimarabidopsis may be congeneric.

Evolutionary processes in the genusCapsella (Brassicaceae)

Capsella comprises diploid (C. grandiflora andC. rubella) and tetraploid taxa. It is argued that the tetraploidC. bursa-pastoris is of intraspecific origin despite disomic inheritance and “fixed heterozygosity”. It is of considerable age as evidenced by the fossil record and molecular data. Gene duplication by polyploidization and a mixed mating system provided the genetic flexibility for greatest colonizing success. Pronounced variation patterns at a micro- and macrogeographic scale are observed inC. bursa-pastoris for many characters including life history traits, leaf morphology and allozymes. This variation pattern can be explained by several components comprising phylogenetic age, random processes, ecotypic variation and colonization history. The adaptive strategy ofC. bursa-pastoris cannot be assigned to either ecotypic differentiation or phenotypic plasticity alone. It depends on the trait under study.

Demographic and random amplified polymorphic DNA analyses reveal high levels of genetic diversity in a clonal violet

We performed demographic and molecular investigations on woodland populations of the clonal herb Viola riviniana in central Germany. We investigated the pattern of seedling recruitment, the amount of genotypic (clonal) variation and the partitioning of genetic variation among and within populations. Our demographic study was carried out in six violet populations of different ages and habitat conditions. It revealed that repeated seedling recruitment takes place in all of these populations, and that clonal propagation is accompanied by high ramet mortality. Our molecular investigations were performed on a subset of three of these six violet populations. Random amplified polymorphic DNA analyses using six primers yielded 45 scorable bands that were used to identify multilocus genotypes, i.e. putative clones. Consistent with our demographic results and independent of population age, we found a large genotypic diversity with a mean proportion of distinguishable genotypes of 0.93 and a mean Simpson’s diversity index of 0.99. Using amova we found a strong genetic differentiation among these violet populations with a ΦST value of 0.41. We suggest that a high selfing rate, limited gene flow due to short seed dispersal distances and drift due to founder effects are responsible for this pattern. Although Viola riviniana is a clonal plant, traits associated with sexual reproduction rather than clonality per se are moulding the pattern of genetic variation in this species.

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana. Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana, showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana, these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species‐specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.

Variation of development time until flowering in natural populations ofCapsella bursa-pastoris (Cruciferae)

Seed samples were collected from wild populations ofCapsella bursa-pastoris along a transsect from Northern to Southern Europe. Progeny was grown in (a) open-field random block experiments (47 populations) and (b) in growth chambers under five to seven controlled temperature regimes (18 populations). Beginning of flowering was recorded, and great differences between and also within populations are documented. Some populations are extremely heterogenous whereas others are homogenous in this respect. Some biotypes react positively when exposed to lower temperatures, others are inhibited. In many cases specific effects of day- and/or night-temperatures can be inferred. In some progenies begin of flowering is independent of temperature as long as this exceeds the 5:10°C regimen. Altogether,Capsella bursa-pastoris displays definite intraspecific variation in time required until flowering. Adaptations to local ecological conditions are obvious. In addition to a genotypic component pronounced environmental interactions provide the plants with a component of phenotypic plasticity. The degree of modificability apparently varies itself and seems to be controlled by selection; the phenotypic plasticity, therefore, displays adaptive variation patterns, too.

Intrageneric phylogeny of Capsella (Brassicaceae) and the origin of the tetraploid C. bursa-pastoris based on chloroplast and nuclear DNA sequences

Polyploidization, often accompanied by hybridization, has been of major importance in flowering plant evolution. Here we investigate the importance of these processes for the evolution of the tetraploid crucifer Capsella bursa-pastoris using DNA sequences from two chloroplast loci as well as from three nuclear low-copy genes. The near-absence of variation at the C. bursa-pastoris chloroplast markers suggests a single and recent origin of the tetraploid. However, despite supporting a single phylogeny, chloroplast data indicate that neither of the extant Capsella diploids is the maternal parent of the tetraploid. Combined with data from the three nuclear loci, our results do not lend support to previous hypotheses on the origin of C. bursa-pastoris as an allopolyploid between the diploids C. grandiflora and C. rubella or an autopolyploid of C. grandiflora. Nevertheless, for each locus, some of the C. bursa-pastoris accessions harbored C. rubella alleles, indicating that C. rubella contributed to the gene pool of C. bursa-pastoris, either through allopolyploid speciation or, more likely, through hybridization and introgression. To our knowledge, this study is the first of a wild, nonmodel plant genus that uses a combination of chloroplast and multiple low-copy nuclear loci for phylogenetic inference of polyploid evolution.