Identification of circular RNAs of Cannabis sativa L. potentially involved in the biosynthesis of cannabinoids.

MAIN CONCLUSION: We identified circRNAs in the Cannabis sativa L. genome and examined their association with 28 cannabinoids in three tissues of C. sativa. Nine circRNAs are potentially involved in the biosynthesis of six cannabinoids. Cannabis sativa L. has been widely used in the production of medicine, textiles, and food for over 2500 years. The main bioactive compounds in C. sativa are cannabinoids, which have multiple important pharmacological actions. Circular RNAs (circRNAs) play essential roles in growth and development, stress resistance, and the biosynthesis of secondary metabolites. However, the circRNAs in C. sativa remain unknown. In this study, to explore the role of circRNAs in cannabinoid biosynthesis, we performed RNA-Seq and metabolomics analysis on the leaves, roots, and stems of C. sativa. We identified 741 overlapping circRNAs by three tools, of which 717, 16, and 8 circRNAs were derived from exonic, intronic, and intergenic, respectively. Functional enrichment analysis indicated that the parental genes (PGs) of circRNAs were enriched in many processes related to biological stress responses. We found that most of the circRNAs showed tissue-specific expression and 65 circRNAs were significantly correlated with their PGs (P < 0.05, |r|≥ 0.5). We also determined 28 cannabinoids by High-performance liquid chromatography-ESI-triple quadrupole-linear ion trap mass spectrometry. Ten circRNAs, including ciR0159, ciR0212, ciR0153, ciR0149, ciR0016, ciR0044, ciR0022, ciR0381, ciR0006, and ciR0025 were found to be associated with six cannabinoids by weighted gene co-expression network analysis. Twenty-nine of 53 candidate circRNAs, including 9 cannabinoids related were validated successfully using PCR amplification and Sanger sequencing. Taken together, all these results would help to enhance our acknowledge of the regulation of circRNAs, and lay the foundation for breeding new C. sativa cultivars with high cannabinoids through manipulating circRNAs.

Genome size and endopolyploidy evolution across the moss phylogeny.

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.

Approaches to integrating genetic data into ecological networks.

As molecular tools for assessing trophic interactions become common, research is increasingly focused on the construction of interaction networks. Here, we demonstrate three key methods for incorporating DNA data into network ecology and discuss analytical considerations using a model consisting of plants, insects, bats and their parasites from the Costa Rica dry forest. The simplest method involves the use of Sanger sequencing to acquire long sequences to validate or refine field identifications, for example of bats and their parasites, where one specimen yields one sequence and one identification. This method can be fully quantified and resolved and these data resemble traditional ecological networks. For more complex taxonomic identifications, we target multiple DNA loci, for example from a seed or fruit pulp sample in faeces. These networks are also well resolved but gene targets vary in resolution and quantification is difficult. Finally, for mixed templates such as faecal contents of insectivorous bats, we use DNA metabarcoding targeting two sequence lengths (157 and 407 bp) of one gene region and a MOTU, BLAST and BIN association approach to resolve nodes. This network type is complex to generate and analyse, and we discuss the implications of this type of resolution on network analysis. Using these data, we construct the first molecular-based network of networks containing 3,304 interactions between 762 nodes of eight trophic functions and involving parasitic, mutualistic and predatory interactions. We provide a comparison of the relative strengths and weaknesses of these data types in network ecology.

DNA barcoding and NMR spectroscopy-based assessment of species adulteration in the raw herbal trade of Saraca asoca (Roxb.) Willd, an important medicinal plant.

Saraca asoca (Roxb.) Willd, commonly known as "Asoka" or "Ashoka," is one of the most important medicinal plants used in raw herbal trade in India. The bark extracts of the tree are used in the treatment of leucorrhea and other uterine disorders besides also having anti-inflammatory, anti-bacterial, anti-pyretic, anti-helminthic, and analgesic activity. The indiscriminate and rampant extraction of the wood to meet the ever-increasing market demand has led to a sharp decline in naturally occurring populations of the species in the country. Consequently, the species has recently been classified as "vulnerable" by the International Union for Conservation of Nature (IUCN). Increasing deforestation and increasing demand for this medicinal plant have resulted in a limited supply and suspected widespread adulteration of the species in the raw herbal trade market. Adulteration is a serious concern due to: (i) reduction in the efficacy of this traditional medicine, (ii) considerable health risk to consumers, and (iii) fraudulent product substitution that impacts the economy for the Natural Health Product (NHP) Industry and consumers. In this paper, we provide the first attempt to assess the extent of adulteration in the raw herbal trade of S. asoca using DNA barcoding validated by NMR spectroscopic techniques. Analyzing market samples drawn from 25 shops, mostly from peninsular India, we show that more than 80 % of the samples were spurious, representing plant material from at least 7 different families. This is the first comprehensive and large-scale study to demonstrate the widespread adulteration of market samples of S. asoca in India. These results pose grave implications for the use of raw herbal drugs, such as that of S. asoca, on consumer health and safety. Based on these findings, we argue for a strong and robust regulatory framework to be put in place, which would ensure the quality of raw herbal trade products and reassure consumer confidence in indigenous medicinal systems. Graphical Abstract DNA barcoding and NMR spectroscopy-based assessment of adulteration in Saraca asoca.

Space-use behaviour of woodland caribou based on a cognitive movement model.

Movement patterns offer a rich source of information on animal behaviour and the ecological significance of landscape attributes. This is especially useful for species occupying remote landscapes where direct behavioural observations are limited. In this study, we fit a mechanistic model of animal cognition and movement to GPS positional data of woodland caribou (Rangifer tarandus caribou; Gmelin 1788) collected over a wide range of ecological conditions. The model explicitly tracks individual animal informational state over space and time, with resulting parameter estimates that have direct cognitive and ecological meaning. Three biotic landscape attributes were hypothesized to motivate caribou movement: forage abundance (dietary digestible biomass), wolf (Canis lupus; Linnaeus, 1758) density and moose (Alces alces; Linnaeus, 1758) habitat. Wolves are the main predator of caribou in this system and moose are their primary prey. Resulting parameter estimates clearly indicated that forage abundance is an important driver of caribou movement patterns, with predator and moose avoidance often having a strong effect, but not for all individuals. From the cognitive perspective, our results support the notion that caribou rely on limited sensory inputs from their surroundings, as well as on long-term spatial memory, to make informed movement decisions. Our study demonstrates how sensory, memory and motion capacities may interact with ecological fitness covariates to influence movement decisions by free-ranging animals.

Assessing product adulteration in natural health products for laxative yielding plants, Cassia, Senna, and Chamaecrista, in Southern India using DNA barcoding.

Medicinal plants such as Cassia, Senna, and Chamaecrista (belonging to the family Fabaceae) are well known for their laxative properties. They are extensively used within indigenous health care systems in India and several other countries. India exports over 5000 metric tonnes per year of these specific herbal products, and the demand for natural health product market is growing at approximately 10-15% annually. The raw plant material used as active ingredients is almost exclusively sourced from wild populations. Consequently, it is widely suspected that the commercial herbal products claiming to contain these species may be adulterated or contaminated. In this study, we have attempted to assess product authentication and the extent of adulteration in the herbal trade of these species using DNA barcoding. Our method includes four common DNA barcode regions: ITS, matK, rbcL, and psbA-trnH. Analysis of market samples revealed considerable adulteration of herbal products: 50% in the case of Senna auriculata, 37% in Senna tora, and 8% in Senna alexandrina. All herbal products containing Cassia fistula were authentic, while the species under the genus Chamaecrista were not in trade. Our results confirm the suspicion that there is rampant herbal product adulteration in Indian markets. DNA barcodes such as that demonstrated in this study could be effectively used as a regulatory tool to control the adulteration of herbal products and contribute to restoring quality assurance and consumer confidence in natural health products.

Understanding the spectacular failure of DNA barcoding in willows (Salix): does this result from a trans-specific selective sweep?

Willows (Salix: Salicaceae) form a major ecological component of Holarctic floras and consequently are an obvious target for a DNA-based identification system. We surveyed two to seven plastid genome regions (~3.8 kb; ~3% of the genome) from 71 Salix species across all five subgenera, to assess their performance as DNA barcode markers. Although Salix has a relatively high level of interspecific hybridization, this may not sufficiently explain the near complete failure of barcoding that we observed: only one species had a unique barcode. We recovered 39 unique haplotypes, from more than 500 specimens, that could be partitioned into six major haplotype groups. A unique variant of group I (haplotype 1*) was shared by 53 species in three of five Salix subgenera. This unusual pattern of haplotype sharing across infrageneric taxa is suggestive of either a massive nonrandom coalescence failure (incomplete lineage sorting), or of repeated plastid capture events, possibly including a historical selective sweep of haplotype 1* across taxonomic sections. The former is unlikely as molecular dating indicates that haplotype 1* originated recently and is nested in the oldest major haplotype group in the genus. Further, we detected significant non-neutrality in the frequency spectrum of mutations in group I, but not outside group I, and demonstrated a striking absence of geographical (isolation by distance) effects in the haplotype distributions of this group. The most likely explanation for the patterns we observed involves recent repeated plastid capture events, aided by widespread hybridization and long-range seed dispersal, but primarily propelled by one or more trans-species selective sweeps.

Genome size evolution in Ontario ferns (Polypodiidae): evolutionary correlations with cell size, spore size, and habitat type and an absence of genome downsizing.

Genome size is known to correlate with a number of traits in angiosperms, but less is known about the phenotypic correlates of genome size in ferns. We explored genome size variation in relation to a suite of morphological and ecological traits in ferns. Thirty-six fern taxa were collected from wild populations in Ontario, Canada. 2C DNA content was measured using flow cytometry. We tested for genome downsizing following polyploidy using a phylogenetic comparative analysis to explore the correlation between 1Cx DNA content and ploidy. There was no compelling evidence for the occurrence of widespread genome downsizing during the evolution of Ontario ferns. The relationship between genome size and 11 morphological and ecological traits was explored using a phylogenetic principal component regression analysis. Genome size was found to be significantly associated with cell size, spore size, spore type, and habitat type. These results are timely as past and recent studies have found conflicting support for the association between ploidy/genome size and spore size in fern polyploid complexes; this study represents the first comparative analysis of the trend across a broad taxonomic group of ferns.

Real-time PCR methods for identification and stability monitoring of Bifidobacterium longum subsp. longum UABl-14 during shelf life.

Bifidobacterium longum subsp. longum UABl-14™ is an important probiotic strain that was found to support digestive health. Here we present the development and validation of real-time PCR methods for strain-specific identification and enumeration of this important strain. The identification method was evaluated for specificity using 22 target samples and 30 non-target samples. All target samples successfully amplified, while no amplification was observed from any non-target samples including other B. longum strains. The identification method was evaluated for sensitivity using three DNA dilution series and the limit of detection was 2 pg. of DNA. Coupled with a viability dye, the method was further validated for quantitative use to enumerate viable cells of UABl-14. The viability dye treatment (PMAxx) was optimized, and a final concentration of 50 µM was found as an effective concentration to inactivate DNA in dead cells from reacting in PCR. The reaction efficiency, linear dynamic range, repeatability, and reproducibility were also evaluated. The reaction efficiency was determined to be 97.2, 95.2, and 95.0% with R2 values of 99%, in three replicates. The linear dynamic range was 1.3 × 102 to 1.3 × 105 genomes. The relative standard deviation (RSD%) for repeatability ranged from 0.03 to 2.80, and for reproducibility ranged from 0.04 to 2.18. The ability of the validated enumeration method to monitor cell counts during shelf life was evaluated by determining the viable counts and total counts of strain UABl-14 in 18 multi-strain finished products. The viable counts were lower than label claims in seven products tested post-expiration and were higher than label claims in products tested pre-expiration, with a slight decrease in viable counts below label claim in three samples that were tested 2-3 months pre-expiration. Interestingly, the total counts of strain UABl-14 were consistently higher than label claims in all 18 products. Thus, the method enables strain-specific stability monitoring in finished products during shelf life, which can be difficult or impossible to achieve using the standard plate count method. The validated methods allow for simultaneous and cost-effective identification and enumeration of strain UABl-14 and represent an advancement in the quality control and quality assurance of probiotics.

Nuclear Magnetic Resonance Fingerprints and Mini DNA Markers for the Authentication of Cinnamon Species Ingredients Used in Food and Natural Health Products.

Cinnamomum verum (syn C. zeylanicum) is considered 'true' cinnamon. However, it is reported that less expensive sources of cinnamon from C. cassia (syn C. aromaticum), C. loureiroi, and C. burmannii (toxic coumarin) may be used in the place of C. verum. We lack the quality assurance tools that are required to differentiate C. verum from other cinnamon species when verifying that the correct species is sourced from ingredient suppliers. The current research on cinnamon species authentication using DNA tools is limited to a few species and the use of high-quality DNA extracted from raw leaf materials. The cinnamon bark traded in the supply chain contains much less DNA and poorer-quality DNA than leaves. Our research advances DNA methods to authenticate cinnamon, as we utilized full-length chloroplast genomes via a genome skimming approach for C. burmannii and C. cassia to facilitate the design of optimal mini DNA markers. Furthermore, we developed and validated the use of NMR fingerprints for several commercial cinnamon species, including the quantification of 16 molecules. NMR fingerprints provided additional data that were useful for quality assessment in cinnamon extract powders and product consistency. Both the new mini DNA markers and NMR fingerprints were tested on commercial cinnamon products.

Flower Species Ingredient Verification Using Orthogonal Molecular Methods.

Flowers are gaining considerable interest among consumers as ingredients in food, beverages, cosmetics, and natural health products. The supply chain trades in multiple forms of botanicals, including fresh whole flowers, which are easier to identify than dried flowers or flowers processed as powdered or liquid extracts. There is a gap in the scientific methods available for the verification of flower species ingredients traded in the supply chains of multiple markets. The objective of this paper is to develop methods for flower species ingredient verification using two orthogonal methods. More specifically, the objectives of this study employed both (1) DNA-based molecular diagnostic methods and (2) NMR metabolite fingerprint methods in the identification of 23 common flower species ingredients. NMR data analysis reveals considerable information on the variation in metabolites present in different flower species, including color variants within species. This study provides a comprehensive comparison of two orthogonal methods for verifying flower species ingredient supply chains to ensure the highest quality products. By thoroughly analyzing the benefits and limitations of each approach, this research offers valuable insights to support quality assurance and improve consumer confidence.

DNA barcoding detects contamination and substitution in North American herbal products.

BACKGROUND: Herbal products available to consumers in the marketplace may be contaminated or substituted with alternative plant species and fillers that are not listed on the labels. According to the World Health Organization, the adulteration of herbal products is a threat to consumer safety. Our research aimed to investigate herbal product integrity and authenticity with the goal of protecting consumers from health risks associated with product substitution and contamination. METHODS: We used DNA barcoding to conduct a blind test of the authenticity for (i) 44 herbal products representing 12 companies and 30 different species of herbs, and (ii) 50 leaf samples collected from 42 herbal species. Our laboratory also assembled the first standard reference material (SRM) herbal barcode library from 100 herbal species of known provenance that were used to identify the unknown herbal products and leaf samples. RESULTS: We recovered DNA barcodes from most herbal products (91%) and all leaf samples (100%), with 95% species resolution using a tiered approach (rbcL + ITS2). Most (59%) of the products tested contained DNA barcodes from plant species not listed on the labels. Although we were able to authenticate almost half (48%) of the products, one-third of these also contained contaminants and or fillers not listed on the label. Product substitution occurred in 30/44 of the products tested and only 2/12 companies had products without any substitution, contamination or fillers. Some of the contaminants we found pose serious health risks to consumers. CONCLUSIONS: Most of the herbal products tested were of poor quality, including considerable product substitution, contamination and use of fillers. These activities dilute the effectiveness of otherwise useful remedies, lowering the perceived value of all related products because of a lack of consumer confidence in them. We suggest that the herbal industry should embrace DNA barcoding for authenticating herbal products through testing of raw materials used in manufacturing products. The use of an SRM DNA herbal barcode library for testing bulk materials could provide a method for 'best practices? in the manufacturing of herbal products. This would provide consumers with safe, high quality herbal products.

Nuclear DNA content variation and evolution in liverworts.

Across embryophytes there is a significant range in DNA content, both in regards to genome size (total DNA in an unreduced chromosome complement) and degree of endoreduplication (when DNA replication not followed by division resulting in various ploidy levels within the same individual). However, there is little information available on DNA content evolution in liverworts, the likely sister group to all other living plants. This study seeks to detect a phylogenetic structure in the variation in genome size and degree of endopolyploidy within liverworts. Furthermore, we test the hypothesis that shifts in breeding systems and genome size are correlated, as polyploidy is suggested to be a possible mechanism for the evolution of monoecy in liverworts and could therefore be associated with larger genome sizes. Genome size was determined for 67 liverwort species from 33 families using flow cytometry. Estimates for 48 species and 16 families are new to science. A phylogeny was reconstructed using the plastid gene rbcL. Over all taxa analyzed, there was a considerable range in genome size estimates with 1C-values from 0.27 pg (Jungermannia rubra) to 20.46 pg (Phyllothallia fuegiana). Large genome sizes were also found in the Haplomitriopsida. None of the liverwort species showed evidence of endopolyploidy. Although some taxa may be polyploids, a correlation between shifts in genome size and breeding system is lacking. Importantly, genome size variation in liverworts exhibits strong phylogenetic signal (Pagel's λ=0.99955).

Genomic valorization of the fine scale classification of small millet landraces in southern India.

Our research seeks to investigate genomic diversity of landraces of millet, addressing a key uncertainty that will provide a framework for (i) a DNA barcode method that could be used for fast, sensitive, and accurate identification of millet landraces, and (ii) millet landrace conservation including biocultural diversity. We found considerable intraspecific variation among 15 landraces representing six species of small millets using nuclear regions (ITS, ITS1, and ITS2); there was no variation in plastid regions (rbcL, matK, and trnH-psbA). An efficacious ITS2 DNA barcode was used to make 100% accurate landrace assignments for 150 blind samples representing 15 landraces. Our research revealed that genomic variation is aligned with a fine-scale classification of landraces using traditional knowledge (TK) of local farmers. The landrace classification was highly correlated with traits (morphological, agricultural, and cultural utility) associated with considerable factors such as yield, drought tolerance, growing season, medicinal properties, and nutrition. This could provide a DNA-based model for conservation of genetic diversity and the associated bicultural diversity (TK) of millet landraces, which has sustained marginal farming communities in harsh environments for many generations.

Lichen conservation in heavily managed boreal forests.

Lichens are an important component of the boreal forest, where they are long lived, tend to accumulate in older stands, and are a major food source for the threatened woodland caribou (Rangifer tarandus caribou). To be fully sustainable, silvicultural practices in the boreal forest must include the conservation of ecological integrity. Dominant forest management practices, however, have short-term negative effects on lichen diversity, particularly the application of herbicides. To better understand the long-term effects of forest management, we examined lichen regeneration in 35 mixed black spruce (Picea mariana) and jack pine (Pinus banksiana) forest stands across northern Ontario to determine recovery following logging and postharvest silvicultural practices. Our forest stands were 25-40 years old and had undergone 3 common sivilcultural treatments that included harvested and planted; harvested, planted, and treated with N-[phosphonomethyl] glycine (glyphosate); and harvested, planted, and treated with 2,4-dichlorophenoxyacetic acid (2,4-D). Forest stands with herbicide treatments had lower lichen biomass and higher beta and gamma diversity than planted stands that were not treated chemically or control stands. In northwestern Ontario, planted stands that were not treated chemically had significantly greater (p < 0.05) alpha diversity than stands treated with herbicides or control stands. Our results show that common silvicultural practices do not emulate natural disturbances caused by wildfires in the boreal forest for the lichen community. We suggest a reduction in the amount of chemical application be considered in areas where lichen biomass is likely to be high and where the recovery of woodland caribou is an objective.

The power of DNA based methods in probiotic authentication.

Introduction: The global probiotic market is growing rapidly, and strict quality control measures are required to ensure probiotic product efficacy and safety. Quality assurance of probiotic products involve confirming the presence of specific probiotic strains, determining the viable cell counts, and confirming the absence of contaminant strains. Third-party evaluation of probiotic quality and label accuracy is recommended for probiotic manufacturers. Following this recommendation, multiple batches of a top selling multi-strain probiotic product were evaluated for label accuracy. Methods: A total of 55 samples (five multi-strain finished products and 50 single-strain raw ingredients) containing a total of 100 probiotic strains were evaluated using a combination of molecular methods including targeted PCR, non-targeted amplicon-based High Throughput Sequencing (HTS), and non-targeted Shotgun Metagenomic Sequencing (SMS). Results: Targeted testing using species-specific or strain-specific PCR methods confirmed the identity of all strains/species. While 40 strains were identified to strain level, 60 strains were identified to species level only due to lack of strain-specific identification methods. In amplicon based HTS, two variable regions of 16S rRNA gene were targeted. Based on V5-V8 region data, ~99% of total reads per sample corresponded to target species, and no undeclared species were detected. Based on V3-V4 region data, ~95%-97% of total reads per sample corresponded to target species, while ~2%-3% of reads matched undeclared species (Proteus species), however, attempts to culture Proteus confirmed that all batches were free from viable Proteus species. Reads from SMS assembled to the genomes of all 10 target strains in all five batches of the finished product. Discussion: While targeted methods enable quick and accurate identification of target taxa in probiotic products, non-targeted methods enable the identification of all species in a product including undeclared species, with the caveats of complexity, high cost, and long time to result.

A multivariate analysis of variation in genome size and endoreduplication in angiosperms reveals strong phylogenetic signal and association with phenotypic traits.

Genome 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 (λ) was strongest for EI in all tissues, and λ 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.

DNA content variation in monilophytes and lycophytes: large genomes that are not endopolyploid.

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.

The effects of rapid desiccation on estimates of plant genome size.

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.