No abstract

In angiosperms, genome size and nucleobase composition (GC content) exhibit pronounced variation with possible adaptive consequences. The hyperdiverse orchid family possessing the unique phenomenon of partial endoreplication (PE) provides a great opportunity to search for interactions of both genomic traits with the evolutionary history of the family.Using flow cytometry, we report values of both genomic traits and the type of endoreplication for 149 orchid species and compare these with a suite of life-history traits and climatic niche data using phylogeny-based statistics. The evolution of genomic traits was further studied using the Brownian motion (BM) and Ornstein-Uhlenbeck (OU) models to access their adaptive potential.Pronounced variation in genome size (341-54 878 Mb), and especially in GC content (23.9-50.5%), was detected among orchids. Diversity in both genomic traits was closely related to the type of endoreplication, plant growth form and climatic conditions. GC content was also associated with the type of dormancy. In all tested scenarios, OU models always outperformed BM models.Unparalleled GC content variation was discovered in orchids, setting new limits for plants. Our study indicates that diversity in both genome size and GC content has adaptive consequences and is tightly linked with evolutionary transitions to PE. † Research 1647Range-wide climatic variation represented by either standard deviations (SD) or median values (med.) of particular Bioclim variables.

Diploids and tetraploids commonly coexist at all spatial scales and exhibit considerable temporal stability in local ploidy mixtures. Mixed-ploidy populations containing fertile triploid hybrids probaby act as effective generators of cytogenetic novelty and may facilitate inter-ploidy gene flow. Neopolyploid mutants were incapable of local establishment.

Whole genome duplication is a major evolutionary event, but its role in ecological divergence remains equivocal. When populations of different ploidy (cytotypes) overlap in space, "contact zones" are formed, allowing the study of evolutionary mechanisms contributing toward ploidy divergence. Multiple contact zones per species' range are often described but rarely leveraged as natural replicates. We explored whether the strength of niche differentiation of diploid and autotetraploid Arabidopsis arenosa varies over distinct contact zones and if the frequency of triploids decreases from seedling to adult stage. METHODS: We characterized ploidy composition and habitat preferences in 264 populations across three contact zones using climatic niche modeling. Ecological differences of cytotypes were also assessed using local vegetation surveys at 110 populations within two contact zones, and at the finer scale within five mixed-ploidy sites. This was complemented by flow cytometry of seedlings. RESULTS: We found no niche differences between diploid and tetraploid populations within contact zones for either climatic or local environmental variables. Comparisons of cytotypes within mixed-ploidy sites found weak niche differences that were inconsistent in direction. Triploid individuals were virtually absent (0.14%) in the field, and they were at a similarly low frequency (0.2%) in ex situ germinated seedlings. CONCLUSIONS: This study demonstrates the strength in investigating different spatial scales across several contact zones when addressing ecological niche differentiation between ploidies. The lack of consistent habitat differentiation of ploidies across the scales and locations supports the recently emerging picture that processes other than ecological differentiation may underlie ploidy coexistence in diploid-autopolyploid systems.

Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.

Best practices in plant cytometry Flow cytometry (FCM) and flow cytometric sorting (FCS) systems have developed as experimental tools of remarkable power and are enjoying an ever-increasing impact in the general field of biology. 1 Application of these tools to plant biology has developed more slowly given that the natural form of plants infrequently resembles that of the single cell suspension, prototypically the hematopoietic system that drove the original development of FCM/FCS. Nevertheless, these systems have had a profound influence at all levels of plant biology, from the study of single cells and subcellular organelles, to the behavior of populations of plants, and ultimately to the performance of ecosystems. It is safe to say their impact has not plateaued, as further applications of this unique technology are increasingly developed by innovative scientists around the world to address questions both in the basic sciences, and to increasingly confront emerging problems in the applied sector. For example, in addressing the challenges of sustainable production of sufficient food resources based on plant breeding involving ploidy-based approaches (e.g., induction of polyploidy) 2 for the needs of our future global citizens, FCM, and FCS systems will play central roles in this effort. The degree to which FCM and FCS systems have impacted plant biology and applied agricultural sciences must not be understated. The major applications of DNA FCM are ploidy level and genome size estimations, and cell cycle analysis/endoreplication (with the later included in a lower percentage of studies). Indeed, FCM is currently/ extensively and almost exclusively employed as the method of choice for measurement of plant genome sizes. 3,4 Measurements of this type impact agriculture in terms of ploidy estimation, with applications ranging from plant biotechnology, breeding and seed quality testing to taxonomy and population biology. They also impact the fundamental plant sciences in terms of biosystematics, ecology, evolution, genomics, and conservation, among other applications. One of the most startling observations of the angiosperms is the bandwidth occupied by genome size, which spans almost 2400-fold. Flow sorting of higher plant chromosomes has provided invaluable information regarding the organization of DNA sequences within plant species. It has also greatly facilitated the process of wholegenome sequencing by permitting subdivision of large genomes into samples comprising entire chromosomes or chromosome arms. 5 FCS methods applied to wall-less cells (protoplasts) expressing fluorescent proteins (FPs) in a cell type-specific manner have allowed elucidation of patterns of co-regulated gene expression and plant hormone gradients identification 6,7 within organized tissues, such as roots. 8,9

Background and Aims Polyploidy has played an important role in the evolution of ferns. However, the dearth of data on cytotype diversity, cytotype distribution patterns and ecology in ferns is striking in comparison with angiosperms and prevents an assessment of whether cytotype coexistence and its mechanisms show similar patterns in both plant groups. Here, an attempt to fill this gap was made using the ploidy-variable and widely distributed Cystopteris fragilis complex. Methods Flow cytometry was used to assess DNA ploidy level and monoploid genome size (Cx value) of 5518 C. fragilis individuals from 449 populations collected over most of the species’ global distributional range, supplemented with data from 405 individuals representing other related species from the complex. Ecological preferences of C. fragilis tetraploids and hexaploids were compared using field-recorded parameters and database-extracted climate data. Key Results Altogether, five different ploidy levels (2x, 4x, 5x, 6x, 8x) were detected and three species exhibited intraspecific ploidy-level variation: C. fragilis, C. alpina and C. diaphana. Two predominant C. fragilis cytotypes, tetraploids and hexaploids, co-occur over most of Europe in a diffuse, mosaic-like pattern. Within this contact zone, 40 % of populations were mixed-ploidy and most also contained pentaploid hybrids. Environmental conditions had only a limited effect on the distribution of cytotypes. Differences were found in the Cx value of tetraploids and hexaploids: between-cytotype divergence was higher in uniform-ploidy than in mixed-ploidy populations. Conclusions High ploidy-level diversity and widespread cytotype coexistence in the C. fragilis complex match the well-documented patterns in some angiosperms. While ploidy coexistence in C. fragilis is not driven by environmental factors, it could be facilitated by the perennial life-form of the species, its reproductive modes and efficient wind dispersal of spores. Independent origins of hexaploids and/or inter-ploidy gene flow may be expected in mixed-ploidy populations according to Cx value comparisons.

Interspecific hybridization, especially when regularly followed by backcrossing (i.e., introgressive hybridization), conveys a substantial risk for many endangered organisms. This is particularly true for narrow endemics occurring within distributional ranges of widespread congeners. An excellent example is provided by the plant genus Knautia (Caprifoliaceae): Locally endemic K. carinthiaca is reported from two isolated populations in southern Austria situated within an area predominantly occupied by widespread K. arvensis. While K. carinthiaca usually inhabits low‐competition communities on rocky outcrops, K. arvensis occurs mainly in dry to mesic managed grasslands, yet both species can coexist in marginal environments and were suspected to hybridize. Flow cytometry revealed that diploid K. carinthiaca only occurs at its locus classicus, whereas the second locality is inhabited by the morphologically similar but tetraploid K. norica. In the, therefore, single population of K. carinthiaca, flow cytometry and AFLP fingerprinting showed signs of introgressive hybridization with diploid K. arvensis. Hybridization patterns were also reflected in intermediate habitat preferences and morphology of the hybrids. Environmental barriers to gene flow seem to prevent genetic erosion of K. carinthiaca individuals from the core ecological niches, restricting most introgressed individuals to peripheral habitats. Efficient conservation of K. carinthiaca will require strict protection of its habitat and ban on forest clear cuts in a buffer zone to prevent invasion of K. arvensis. We demonstrate the large potential of multidisciplinary approaches combining molecular, cytometric, and ecological tools for a reliable inventory and threat assessment of rare species.