Agroecosystems are dynamic systems that experience frequent chemical inputs and changes in plant cover. The objective of this study was to test whether abiotic (soil chemical properties and climate) and biotic (plant host identity) factors influence the spatial and temporal structuring of arbuscular mycorrhizal fungal (AMF) communities in a semi-arid prairie agroecosystem. 454 GS FLX+ high-throughput sequencing technology was successfully utilized to characterize the AMF communities based on long reads (mean length: 751.7 bp) and generated high-resolution data with excellent taxonomic coverage. The composition of the AMF community colonizing roots of the three crops (pea, lentil, and wheat) significantly differed, but plant host identity had a minimal effect on the composition of the AMF community in the soil. We observed a temporal shift in the composition of AMF communities in the roots and surrounding soil of the crops during the growing season. This temporal shift was particularly evident in the root-associated AMF community and was correlated with soil phosphate flux and climatic variables. In contrast, the spatial structuring of the AMF community in the site was correlated with soil pH and electrical conductivity. Individual AMF taxa were significantly correlated with pH, electrical conductivity, and phosphate flux, and these relationships were phylogenetically conserved at the genus level within the Glomeromycota.

Less than 1% of known monilophytes and lycophytes have a genome size estimate, and substantially less is known about the presence and prevalence of endopolyploid nuclei in these groups. Thirty-one monilophyte species (including three horsetails) and six lycophyte species were collected in Ontario, Canada. Using flow cytometry, genome size and degree of endopolyploidy were estimated for 37 species. Across the five orders covered, 1Cx-values averaged 4.2 pg in the Lycopodiales, 18.1 pg for the Equisetales, 5.06 pg for a single representative of the Ophioglossales, 14.3 pg for the Osmundales, and 7.06 pg for the Polypodiales. There was no indication of endoreduplication in any of the leaf, stem, or root tissue analyzed. This information is essential to our understanding of DNA content evolution in land plants.

Flow cytometry has become the dominant method for estimating nuclear DNA content in plants, either for ploidy determination or quantification of absolute genome size. Current best practices for flow cytometry involve the analysis of fresh tissue, however, this imposes significant limitations on the geographic scope and taxonomic diversity of plants that can be included in large-scale genome size studies. Dried tissue has been used increasingly in recent years, but largely in the context of ploidy analysis. Here we test rapid tissue drying with silica gel as a method for use in genome size studies, potentially enabling broader geographic sampling of plants when fresh tissue collection is not feasible. Our results indicate that rapid drying introduces comparatively minor error (<10%), which is similar to the error introduced by other common methodological variations such as instrument. Additionally, the relative effect of drying on genome size and data quality varied between species and buffers. Tissue desiccation provides a promising approach for expanding our knowledge of plant genome size diversity.

Flow cytometry (FCM) is currently the most widely‐used method to establish nuclear DNA content in plants. Since simple, 1‐3‐parameter, flow cytometers, which are sufficient for most plant applications, are commercially available at a reasonable price, the number of laboratories equipped with these instruments, and consequently new FCM users, has greatly increased over the last decade. This paper meets an urgent need for comprehensive recommendations for best practices in FCM for different plant science applications. We discuss advantages and limitations of establishing plant ploidy, genome size, DNA base composition, cell cycle activity, and level of endoreduplication. Applications of such measurements in plant systematics, ecology, molecular biology research, reproduction biology, tissue cultures, plant breeding, and seed sciences are described. Advice is included on how to obtain accurate and reliable results, as well as how to manage troubleshooting that may occur during sample preparation, cytometric measurements, and data handling. Each section is followed by best practice recommendations; tips as to what specific information should be provided in FCM papers are also provided.

SummaryGenome size (C-value) and endopolyploidy (endoreduplication index, EI) are known to correlate with various morphological and ecological traits, in addition to phylogenetic placement. A phylogenetically controlled multivariate analysis was used to explore the relationships between DNA content and phenotype in angiosperms.Seeds from 41 angiosperm species (17 families) were grown in a common glasshouse experiment. Genome size (2C-value and 1Cx-value) and EI (in four tissues: leaf, stem, root, petal) were determined using flow cytometry. The phylogenetic signal was calculated for each measure of DNA content, and phylogenetic canonical correlation analysis (PCCA) explored how the variation in genome size and EI was correlated with 18 morphological and ecological traits.Phylogenetic signal (k) was strongest for EI in all tissues, and k was stronger for the 2C-value than the 1Cx-value. PCCA revealed that EI was correlated with pollen length, stem height, seed mass, dispersal mechanism, arbuscular mycorrhizal association, life history and flowering time, and EI and genome size were both correlated with stem height and life history.PCCA provided an effective way to explore multiple factors of DNA content variation and phenotypic traits in a phylogenetic context. Traits that were correlated significantly with DNA content were linked to plant competitive ability.

Background and Aims Compared with other plant lineages, bryophytes have very small genomes with little variation across species, and high levels of endopolyploid nuclei. This study is the first analysis of moss genome evolution over a broad taxonomic sampling using phylogenetic comparative methods. We aim to determine whether genome size evolution is unidirectional as well as examine whether genome size and endopolyploidy are correlated in mosses. Methods Genome size and endoreduplication index (EI) estimates were newly generated using flow cytometry from moss samples collected in Canada. Phylogenetic relationships between moss species were reconstructed using GenBank sequence data and maximum likelihood methods. Additional 1C-values were compiled from the literature and genome size and EI were mapped onto the phylogeny to reconstruct ancestral character states, test for phylogenetic signal and perform phylogenetic independent contrasts. Key Results Genome size and EI were obtained for over 50 moss taxa. New genome size estimates are reported for 33 moss species and new EIs are reported for 20 species. In combination with data from the literature, genome sizes were mapped onto a phylogeny for 173 moss species with this analysis, indicating that genome size evolution in mosses does not appear to be unidirectional. Significant phylogenetic signal was detected for genome size when evaluated across the phylogeny, whereas phylogenetic signal was not detected for EI. Genome size and EI were not found to be significantly correlated when using phylogenetically corrected values. Conclusions Significant phylogenetic signal indicates closely related mosses have more similar genome sizes and EI values. This study supports that DNA content in mosses is defined by small genomes that are highly endopolyploid, suggesting strong selective pressure to maintain these features. Further research is needed to understand the functional significance of DNA content evolution in mosses.

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Endopolyploidy occurs when DNA replication is not followed by mitotic nuclear division, resulting in tissues or organisms with nuclei of varying ploidy levels. Endopolyploidy appears to be a common phenomenon in plants, though the prevalence of endopolyploidy has not been determined in bryophytes (including mosses and liverworts). Forty moss species and six liverwort species were analyzed for the degree of endopolyploidy using flow cytometry. Nuclei were extracted in LB01 buffer and stained with propidium iodide. Of the forty moss species, all exhibited endopolyploid nuclei (mean cycle value=0.65±0.038) except for theSphagnummosses (mean cycle value=0). None of the liverwort species had endopolyploid nuclei (mean cycle value= 0.04 ± 0.014). As bryophytes form a paraphyletic grade leading to the tracheophytes, understanding the prevalence and role of endopolyploidy in this group is important.