Calochortus gunnisonii furthers evidence for the complex genetic legacy of historical climate change in the southern Rocky Mountains.

PREMISE OF THE STUDY: Climate cycles of the Quaternary have impacted plants at a global scale, leaving behind a complex genetic legacy. Species of the northern Rocky Mountains of North America were exposed to more uniform glacial patterns than the central and southern ranges, where synergistic relationships between temperature and precipitation caused differences in the timing and extent of glacier onset. We examined the genetic impacts of climate oscillations on Calochortus gunnisonii (Liliaceae) in the central and southern Rocky Mountains. METHODS: Populations were sampled from disjunct mountain ranges across the basins of Wyoming and northern and central Colorado. Allelic data from nuclear microsatellites and plastid sequences (trnV-ndhC, petA-psbJ, and rpl16) were used to examine patterns of genetic structure between and among populations along the southern Rocky Mountain corridor. KEY RESULTS: We infer considerable population structure concordant with mountain range of origin. Clustering analysis supports separate north and south genetic clusters on either side of major basins in Wyoming, suggesting that populations were maintained in two distinct refugia. Additionally, populations within the Sierra Madre Range of southern Wyoming show localized, divergent genetic signal indicative of a third potential glacial refugium. By contrast, recent genetic admixture is observed in the Laramie, Medicine Bow, and Front ranges, where population expansion from glacial refugia has likely occurred. CONCLUSIONS: We conclude that during climate cycles of the Quaternary, C. gunnisonii experienced periods of population expansion and reduction, habitat fragmentation, isolation in three or more refugia, and admixture mirroring genetic impacts of other southern Rocky Mountains organisms.

Phylogeography and genetic structure of endemic Acmispon argophyllus and A. dendroideus (Fabaceae) across the California Channel Islands.

PREMISE OF THE STUDY: Taxa inhabiting the California Channel Islands exhibit variation in their degree of isolation, but few studies have considered patterns across the entire archipelago. We studied phylogeography of insular Acmispon argophyllus and A. dendroideus to determine whether infraspecific taxa are genetically divergent and to elucidate patterns of diversification across these islands. METHODS: DNA sequences were collected from nuclear (ADH) and plastid genomes (rpL16, ndhA, psbD-trnT) from >450 samples on the Channel Islands and California. We estimated population genetic diversity and structure, phylogenetic patterns among populations, and migration rates, and tested for population growth. KEY RESULTS: Populations of northern island A. argophyllus var. niveus are genetically distinct from conspecific populations on southern islands. On the southern islands, A. argophyllus var. argenteus populations on Santa Catalina are phylogenetically distinct from populations of var. argenteus and var. adsurgens on the other southern islands. For A. dendroideus, we found the varieties to be monophyletic. Populations of A. dendroideus var. traskiae on San Clemente are genetically differentiated from other conspecific populations, whereas populations on the northern islands and Santa Catalina show varying degrees of gene flow. Evidence of population growth was found in both species. CONCLUSIONS: Oceanic barriers between islands have had a strong influence on population genetic structure in both Acmispon species, although the species have differing phylogeographic patterns. This study provides a contrasting pattern of dispersal on a near island system that does not follow a strict stepping-stone model, commonly found on isolated island systems.

Morphological and genetic discrepancies in populations of Oreocarya paradoxa and O. revealii: the impact of edaphic selection on recent diversification in the Colorado Plateau.

PREMISE OF THE STUDY: Investigations of recently derived and edaphically (soil) defined plant systems have provided insight into important mechanisms of ecological divergence. We investigated the impact of edaphic adaptation on recent divergence between two Colorado Plateau endemics: the gypsum facultative Oreocarya revealii (Boraginaceae) and its more generalist sister species O. paradoxa. We assessed morphological stability, genetic identity, and soil chemistry to determine whether O. revealii is a distinct lineage edaphically adapted from O. paradoxa, as has been described in the literature. METHODS: We genotyped 21 populations throughout the ranges of both species using 11 microsatellite markers and three plastid regions (trnL-F, trnT-L, trnQ-rps16) for haplotype analysis. We compared these data with soil chemistry (Ca and S concentrations, indicating gypsum levels), location, and morphological identity of populations. KEY RESULTS: Soil chemistry failed to explain genetic or morphological identity in either taxon. Haplotype analysis suggests ancestral variation in the more geographically restricted O. revealii, along with regional geographic isolation. A discontinuity was identified between the morphological and genetic identity in several populations, suggesting incomplete lineage sorting and the nonfixation of identifying morphological traits. CONCLUSIONS: Oreocarya revealii is unlikely to have arisen via edaphic selection, because soil chemistry of population sites, morphology of individuals, and genetic identity are not strongly correlated. The nonfixation of identifying traits is likely a result of recent divergence in this system, and the potentiality of such discrepancies should be considered when investigating recently diversified gypsum-associated groups.

Uncomfortably high: Testing reveals inflated THC potency on retail Cannabis labels.

Legal Cannabis products in the United States are required to report THC potency (total THC % by dry weight) on packaging, however concerns have been raised that reported THC potency values are inaccurate. Multiple studies have demonstrated that THC potency is a primary factor in determining pricing for Cannabis flower, so it has an outsized role in the marketplace. Reports of inflated THC potency and "lab shopping" to obtain higher THC potency results have been circulating for some time, but a side-by-side investigation of the reported potency and flower in the package has not previously been conducted. Using HPLC, we analyzed THC potency in 23 samples from 10 dispensaries throughout the Colorado Front Range and compared the results to the THC potency reported on the packaging. Average observed THC potency was 14.98 +/- 2.23%, which is substantially lower than recent reports summarizing dispensary reported THC potency. The average observed THC potency was 23.1% lower than the lowest label reported values and 35.6% lower than the highest label reported values. Overall, ~70% of the samples were more than 15% lower than the THC potency numbers reported on the label, with three samples having only one half of the reported maximum THC potency. Although the exact source of the discrepancies is difficult to determine, a lack of standardized testing protocols, limited regulatory oversight, and financial incentives to market high THC potency likely play a significant role. Given our results it is urgent that steps are taken to increase label accuracy of Cannabis being sold to the public. The lack of accurate reporting of THC potency can have impacts on medical patients controlling dosage, recreational consumers expecting an effect aligned with price, and trust in the industry as a whole. As the legal cannabis market continues to grow, it is essential that the industry moves toward selling products with more accurate labeling.

Variable length chloroplast markers for population genetic studies in Acmispon (Fabaceae).

PREMISE OF THE STUDY: To estimate genetic structure, chloroplast loci containing length-variable regions were developed for two legumes, Acmispon argophyllus and A. dendroideus. • METHODS AND RESULTS: Primers for 14 chloroplast loci containing repeat regions were developed from the chloroplast genome sequence of the legume Lotus japonicus and tested in Acmispon. Nine loci exhibited polymorphism in Acmispon, with up to six alleles per locus. Gene diversity ranged from 0 to 0.775 in A. argophyllus and 0.142 to 0.766 in A. dendroideus. The primers also amplified in other Acmispon species. Sequencing of the fragments revealed discordance between fragment sizes and underlying sequence for three loci containing complex repeat regions. • CONCLUSIONS: Although genotypes were easily generated and sized, sequencing may be more informative of genetic variation in loci with complex repeat regions. These loci exhibit substantial variation and should be useful for understanding genetic structure associated with seed dispersal in Acmispon.

Human olfactory discrimination of genetic variation within Cannabis strains.

Cannabis sativa L. is grown and marketed under a large number of named strains. Strains are often associated with phenotypic traits of interest to consumers, such as aroma and cannabinoid content. Yet genetic inconsistencies have been noted within named strains. We asked whether genetically inconsistent samples of a commercial strain also display inconsistent aroma profiles. We genotyped 32 samples using variable microsatellite regions to determine a consensus strain genotype and identify genetic outliers (if any) for four strains. Results were used to select 15 samples for olfactory testing. A genetic outlier sample was available for all but one strain. Aroma profiles were obtained by 55 sniff panelists using quantitative sensory evaluation of 40 odor descriptors. Within a strain, aroma descriptor frequencies for the genetic outlier were frequently at odds with those of the consensus samples. It appears that within-strain genetic differences are associated with differences in aroma profile. Because these differences were perceptible to untrained panelists, they may also be noticed by retail consumers. Our results could help the cannabis industry achieve better control of product consistency.

Evolutionary diversification and geographical isolation in Dubautia laxa (Asteraceae), a widespread member of the Hawaiian silversword alliance.

BACKGROUND AND AIMS: The Hawaiian silversword alliance (Asteraceae) is one the best examples of a plant adaptive radiation, exhibiting extensive morphological and ecological diversity. No research within this group has addressed the role of geographical isolation, independent of ecological adaptation, in contributing to taxonomic diversity. The aims of this study were to examine genetic differentiation among subspecies of Dubautia laxa (Asteraceae) to determine if allopatric or sympatric populations and subspecies form distinct genetic clusters to understand better the role of geography in diversification within the alliance. METHODS: Dubautia laxa is a widespread member of the Hawaiian silversword alliance, occurring on four of the five major islands of the Hawaiian archipelago, with four subspecies recognized on the basis of morphological, ecological and geographical variation. Nuclear microsatellites and plastid DNA sequence data were examined. Data were analysed using maximum-likelihood and Bayesian phylogenetic methodologies to identify unique evolutionary lineages. KEY RESULTS: Plastid DNA sequence data resolved two highly divergent lineages, recognized as the Laxa and Hirsuta groups, that are more similar to other members of the Hawaiian silversword alliance than they are to each other. The Laxa group is basal to the young island species of Dubautia, whereas the Hirsuta group forms a clade with the old island lineages of Dubautia and with Argyroxiphium. The divergence between the plastid groups is supported by Bayesian microsatellite clustering analyses, but the degree of nuclear differentiation is not as great. Clear genetic differentiation is only observed between allopatric populations, both within and among islands. CONCLUSIONS: These results indicate that geographical separation has aided diversification in D. laxa, whereas ecologically associated morphological differences are not associated with neutral genetic differentiation. This suggests that, despite the stunning ecological adaptation observed, geography has also played an important role in the Hawaiian silversword alliance plant adaptive radiation.

Microsatellite primers for the narrowly endemic shrub Eriogonum giganteum (Polygonaceae).

PREMISE OF THE STUDY: Microsatellite primers were designed for Eriogonum giganteum var. formosum, an endemic shrub of San Clemente Island, to investigate population structure, genetic diversity, and demographic history. METHODS AND RESULTS: Twelve polymorphic microsatellite loci were isolated from the California Channel Island endemic Eriogonum and were screened for variability. The primers amplified one to eight alleles in the target taxon. Many primers also amplified in conspecific and congeneric (E. arborescens, E. fasciculatum, E. grande, E. latifolium, and E. parvifolium) taxa and in the closely related Chorizanthe valida. The total number of alleles per locus for all taxa screened ranged from three to 24. CONCLUSIONS: These primers will be useful for conservation genetic and evolutionary studies within the California Channel Island endemic Eriogonum.

Comparative Genetic Structure of Cannabis sativa Including Federally Produced, Wild Collected, and Cultivated Samples.

Currently in the United States, the sole licensed facility to cultivate Cannabis sativa L. for research purposes is the University of Mississippi, which is funded by the National Institute on Drug Abuse (NIDA). Studies researching Cannabis flower consumption rely on NIDA-supplied "research grade marijuana." Previous research found that cannabinoid levels of NIDA-supplied Cannabis do not align with commercially available Cannabis. We sought to investigate the genetic identity of Cannabis supplied by NIDA relative to common categories within the species. This is the first genetic study to include "research grade marijuana" from NIDA. Samples (49) were assigned as Wild Hemp (feral; 6) and Cultivated Hemp (3), NIDA (2), CBD drug type (3), and high THC drug type subdivided into Sativa (11), Hybrid (14), and Indica (10). Ten microsatellites targeting neutral non-coding regions were used. Clustering and genetic distance analyses support a division between hemp and drug-type Cannabis. All hemp samples clustered genetically, but no clear distinction of Sativa, Hybrid, and Indica subcategories within retail marijuana samples was found. Interestingly, the two analyzed "research grade marijuana" samples obtained from NIDA were genetically distinct from most drug-type Cannabis available from retail dispensaries. Although the sample size was small, "research grade marijuana" provided for research is genetically distinct from most retail drug-type Cannabis that patients and patrons are consuming.

Genomic Evidence That Governmentally Produced Cannabis sativa Poorly Represents Genetic Variation Available in State Markets.

The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genomes of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDA's varieties differ from widely available medical, recreational, or industrial Cannabis. Furthermore, our results suggest that NIDA's varieties lack diversity in the single-copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome. Therefore, results based on NIDA's varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation.

Genetic tools weed out misconceptions of strain reliability in Cannabis sativa: implications for a budding industry.

BACKGROUND: Unlike other plants, Cannabis sativa is excluded from regulation by the United States Department of Agriculture (USDA). Distinctive Cannabis varieties are ostracized from registration and therefore nearly impossible to verify. As Cannabis has become legal for medical and recreational consumption in many states, consumers have been exposed to a wave of novel Cannabis products with many distinctive names. Despite more than 2000 named strains being available to consumers, questions about the consistency of commercially available strains have not been investigated through scientific methodologies. As Cannabis legalization and consumption increases, the need to provide consumers with consistent products becomes more pressing. In this research, we examined commercially available, drug-type Cannabis strains using genetic methods to determine if the commonly referenced distinctions are supported and if samples with the same strain name are consistent when obtained from different facilities. METHODS: We developed ten de-novo microsatellite markers using the "Purple Kush" genome to investigate potential genetic variation within 30 strains obtained from dispensaries in three states. Samples were examined to determine if there is any genetic distinction separating the commonly referenced Sativa, Indica and Hybrid types and if there is consistent genetic identity found within strain accessions obtained from different facilities. RESULTS: Although there was strong statistical support dividing the samples into two genetic groups, the groups did not correspond to commonly reported Sativa/Hybrid/Indica types. The analyses revealed genetic inconsistencies within strains, with most strains containing at least one genetic outlier. However, after the removal of obvious outliers, many strains showed considerable genetic stability. CONCLUSIONS: We failed to find clear genetic support for commonly referenced Sativa, Indica and Hybrid types as described in online databases. Significant genetic differences within samples of the same strain were observed indicating that consumers could be provided inconsistent products. These differences have the potential to lead to phenotypic differences and unexpected effects, which could be surprising for the recreational user, but have more serious implications for patients relying on strains that alleviate specific medical symptoms.

DNA barcodes from snake venom: a broadly applicable method for extraction of DNA from snake venoms.

DNA barcoding is a simple technique used to develop a large-scale system of classification that is broadly applicable across a wide variety of taxa. DNA-based analysis of snake venoms can provide a system of classification independent of currently accepted taxonomic relationships by generating DNA barcodes specific to each venom sample. DNA purification from dried snake venoms has previously required large amounts of starting material, has resulted in low yields and inconsistent amplification, and was possible with front-fanged snakes only. Here, we present a modified DNA extraction protocol applied to venoms of both front- and rear-fanged snakes that requires significantly less starting material (1 mg) and yields sufficient amounts of DNA for successful PCR amplification of regions commonly used for DNA barcoding. [Formula: see text].

Ecological speciation in the East Maui-endemic Dubautia (Asteraceae) species.

Adaptive diversification can be viewed as a balance between the conservative force of interpopulation gene flow and selection for differential environments. In this paper, we examine ecological, morphological, and genetic differentiation in a small clade consisting of four East Maui-endemic species of Dubautia: D. menziesii, D. platyphylla, D. reticulata, and D. waianapanapaensis, in the Hawaiian silversword alliance (Asteraceae). The East Maui clade is apparently recently derived (less than 1 million years ago) and is geographically restricted yet displays significant ecological and morphological differences. We used geographic data from historical herbarium specimens, measurements of plant architecture and leaf morphometrics, and measures of genetic differentiation in both microsatellite and nuclear coding loci to examine the correlation of different forms of divergence in this small species flock. We found overlap in large-scale geographic distributions, significant differentiation in most habitat factors, significant microsatellite differentiation, and many shared alleles at nuclear coding loci suggesting on-going lineage sorting. Despite the presence of apparent hybrids in some populations, microsatellite variation is consistent with isolation among species. Using Mantel tests, we compared the direction and extent of diversification among different datasets, to determine whether ecological/morphological divergence was correlated with genetic divergence. Correlations among different datasets showed that habitat was strongly correlated with plant architecture but not leaf morphology. Taken together, these results indicate that ecological and morphological diversification has driven genetic divergence at rapidly evolving microsatellite loci, whereas there is continuing lineage sorting at neutral sites in nuclear coding loci.

Characterization of 13 microsatellite markers for Calochortus gunnisonii (Liliaceae) from Illumina MiSeq sequencing.

PREMISE OF THE STUDY: Microsatellite primers were designed for Calochortus gunnisonii (Liliaceae), a montane lily species of the central and southern Rocky Mountains, using next-generation DNA sequencing. The markers will be used to investigate population structure, genetic diversity, and demographic history. METHODS AND RESULTS: Thirteen polymorphic microsatellite loci were isolated from C. gunnisonii using Illumina MiSeq next-generation DNA sequencing and bioinformatic screening. The mean number of alleles per locus ranged from 4.15 to 5.92 (avg. = 4.97). Observed and expected heterozygosity ranged from 0.077 to 0.871 and 0.213 to 0.782, respectively. The primers were also tested for cross-species amplification value with C. flexuosus, C. nuttallii, C. kennedyi var. kennedyi, and C. subalpinus. CONCLUSIONS: These primers will be useful for genetic and evolutionary studies across C. gunnisonii's range within the southern and central Rocky Mountains. Furthermore, these markers have proven valuable for cross-species amplifications within Calochortus.

Isolation of microsatellite loci from the endangered plant Galium catalinense subspecies acrispum (Rubiaceae).

Galium catalinense subspecies acrispum (Rubiaceae) is a state-endangered perennial shrub endemic to San Clemente Island. Eight polymorphic microsatellite loci were isolated from G. catalinense ssp. acrispum. These loci show high levels of variability, averaging 6.5 alleles per locus and an expected heterozygosity of 0.550. One locus exhibited significant deviations from Hardy-Weinberg equilibrium (P < 0.01) and one pair of loci exhibited significant linkage disequilibrium.

PERMANENT GENETIC RESOURCES: Isolation of microsatellite loci from the endangered plant Sibara filifolia (Brassicaceae).

Sibara filifolia (Brassicaceae) is a federally endangered annual herb found on two of the California Channel Islands. Previous studies based on allozymes revealed little genetic variability on San Clemente Island. Nine polymorphic microsatellite loci were isolated from individuals on San Clemente Island. We found low levels of allelic variation (mean N(A)  = 2.3), with seven loci exhibiting significant deviations from Hardy-Weinberg equilibrium (P < 0.01) and 10 pairs of loci exhibiting significant linkage disequilibrium (P < 0.01). Most of the observed variability (mean H(O)  = 0.003) occurred among populations or in rare homozygous individuals.