Deciphering the evolution of herbicide resistance in weeds.

Resistance to herbicides in arable weeds is increasing rapidly worldwide and threatening global food security. Resistance has now been reported to all major herbicide modes of action despite the development of resistance management strategies in the 1990s. We review here recent advances in understanding the genetic bases and evolutionary drivers of herbicide resistance that highlight the complex nature of selection for this adaptive trait. Whereas early studied cases of resistance were highly herbicide-specific and largely under monogenic control, cases of greatest concern today generally involve resistance to multiple modes of action, are under polygenic control, and are derived from pre-existing stress response pathways. Although 'omics' approaches should enable unraveling the genetic bases of complex resistances, the appearance, selection, and spread of herbicide resistance in weed populations can only be fully elucidated by focusing on evolutionary dynamics and implementing integrative modeling efforts.

De novo genome assembly of the economically important weed horseweed using integrated data from multiple sequencing platforms.

Horseweed (Conyza canadensis), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n = 2x = 18), with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic bases of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000, and PacBio RS) using various libraries with different insertion sizes (approximately 350 bp, 600 bp, 3 kb, and 10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (approximately 350× coverage) of data, the genome was assembled into 13,966 scaffolds with 50% of the assembly = 33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (approximately 153 kb) and a nearly complete mitochondrial genome (approximately 450 kb in 120 scaffolds). The nuclear genome is composed of 44,592 protein-coding genes. Genome resequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single-nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or -susceptible biotypes. The draft genome will be useful to better understand weediness and the evolution of herbicide resistance and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this article represents the first published draft genome of an agricultural weed.

Small population size limits reproduction in an invasive grass through both demography and genetics.

Small populations of founding individuals or survivors of incomplete management programs often represent critical transitions in biological invasions. Theory predicts that population size affects reproduction and, consequently, a population's expansion, but there are few empirical tests, and fewer that account for the reduced genetic diversity that often accompanies small population size. We created experimental small populations of invasive ryegrass (Lolium multiflorum) with population size varying independently from genetic diversity. Treatment independence was achieved by cloning plants to increase population size without changing diversity. Plant fitness was measured as the proportion of florets producing a seed. We analyzed the effects of population size, genetic diversity, and their interaction using ANCOVAs, one of which accounted for variation in individual plant growth. As predicted, smaller populations produced significantly lower proportion seed set. Low genetic diversity also reduced seed set, but this was best interpreted as part of a significant interaction with population size. Specifically, the effect of population size on the proportion seed set was over five times larger for populations in the medium genetic diversity treatment than the highest diversity treatment, and 6.7 times larger for populations with the lowest level of diversity. Population size variation had biologically meaningful consequences, as the rate of seed set within the low diversity treatment increased by 80 % with increasing population size. The results indicate that both the demographics and genetics of populations can influence reproduction and invasive potential, and must be considered when assessing risk and designing management plans for invasive plants.

A molecular phylogenetic analysis of invasive and ornamental brooms and their relationships within the Genistoid legumes.

The Cytisus-Genista complex includes species that have become invasive following introduction into new geographic ranges as ornamental shrubs. Despite their impacts, the evolutionary relationships among invasives, ornamentals, and native-range species have never been investigated. Our objective was to examine relationships within the Cytisus-Genista complex to determine (1) the taxonomic identity of invasive "French broom" and ornamental "sweet broom" and (2) whether "sweet broom" contributes to "French broom" populations directly or via hybridization. We used sequence data from chloroplast and nuclear regions to gain insight into evolutionary origins and to confirm taxonomic status. Our phylogenetic analyses suggest a complex evolutionary history that includes hybridization events. Placement of invasive and ornamental individuals within the Cytisus-Genista complex resolves taxonomic uncertainty in these groups, as our phylogenetic analyses recovered separate "French broom" and "sweet broom" clades within the G. monspessulana clade in the genus Genista. Extensive cloning and sequencing of the ITS region revealed that, although the majority of invasive "French broom" in California is Genista monspessulana, hybridization with individuals from the ornamental "sweet broom" clade likely occurs in populations throughout the state.

Increased temperatures and elevated CO2 levels reduce the sensitivity of Conyza canadensis and Chenopodium album to glyphosate.

Herbicides are the most commonly used means of controlling weeds. Recently, there has been growing concern over the potential impacts of global climate change, specifically, increasing temperatures and elevated carbon dioxide (CO2) concentrations, on the sensitivity of weeds to herbicides. Here, glyphosate response of both Conyza canadensis and Chenopodium album was evaluated under different environmental conditions. Reduced glyphosate sensitivity was observed in both species in response to increased temperature, elevated CO2 level, and the combination of both factors. Increased temperature had greater effect on plant survival than elevated CO2 level. In combination, high temperature and elevated CO2 level resulted in loss of apical dominance and rapid necrosis in glyphosate-treated plants. To investigate the mechanistic basis of reduced glyphosate sensitivity, translocation was examined using 14C-glyphosate. In plants that were subjected to high temperatures and elevated CO2 level, glyphosate was more rapidly translocated out of the treated leaf to shoot meristems and roots than in plants grown under control conditions. These results suggest that altered glyphosate translocation and tissue-specific sequestration may be the basis of reduced plant sensitivity. Therefore, overreliance on glyphosate for weed control under changing climatic conditions may result in more weed control failures.

Multiple resistance to glyphosate, paraquat and ACCase-inhibiting herbicides in Italian ryegrass populations from California: confirmation and mechanisms of resistance.

BACKGROUND: Glyphosate, paraquat and acetyl CoA carboxylase (ACCase)-inhibiting herbicides are widely used in California annual and perennial cropping systems. Recently, glyphosate, paraquat, and ACCase- and acetolactate synthase (ALS)-inhibitor resistance was confirmed in several Italian ryegrass populations from the Central Valley of California. This research characterized the possible mechanisms of resistance. RESULTS: Multiple-resistant populations (MR1, MR2) are resistant to several herbicides from at least three modes of action. Dose-response experiments revealed that the MR1 population was 45.9-, 122.7- and 20.5-fold, and the MR2 population was 24.8-, 93.9- and 4.0-fold less susceptible to glyphosate, sethoxydim and paraquat, respectively, than the susceptible (Sus) population. Accumulation of shikimate in Sus plants was significantly greater than in MR plants 32 h after light pretreatments. Glyphosate resistance in MR plants was at least partially due to Pro106-to-Ala and Pro106-to-Thr substitutions at site 106 of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS gene copy number and expression level were similar in plants from the Sus and MR populations. An Ile1781-to-Leu substitution in ACCase gene of MR plants conferred a high level of resistance to sethoxydim and cross-resistance to other ACCase-inhibitors. Radiolabeled herbicide studies and phosphorimaging indicated that MR plants had restricted translocation of 14 C-paraquat to untreated leaves compared to Sus plants. CONCLUSION: This study shows that multiple herbicide resistance in Italian ryegrass populations in California, USA, is due to both target-site and non-target-site resistance mechanisms. © 2017 Society of Chemical Industry.

The power and potential of genomics in weed biology and management.

There have been previous calls for, and efforts focused on, realizing the power and potential of weed genomics for better understanding of weeds. Sustained advances in genome sequencing and assembly technologies now make it possible for individual research groups to generate reference genomes for multiple weed species at reasonable costs. Here, we present the outcomes from several meetings, discussions, and workshops focused on establishing an International Weed Genomics Consortium (IWGC) for a coordinated international effort in weed genomics. We review the 'state of the art' in genomics and weed genomics, including technologies, applications, and on-going weed genome projects. We also report the outcomes from a workshop and a global survey of the weed science community to identify priority species, key biological questions, and weed management applications that can be addressed through greater availability of, and access to, genomic resources. Major focus areas include the evolution of herbicide resistance and weedy traits, the development of molecular diagnostics, and the identification of novel targets and approaches for weed management. There is increasing interest in, and need for, weed genomics, and the establishment of the IWGC will provide the necessary global platform for communication and coordination of weed genomics research. © 2018 Society of Chemical Industry.

Nucleotide Diversity at Site 106 of EPSPS in Lolium perenne L. ssp. multiflorum from California Indicates Multiple Evolutionary Origins of Herbicide Resistance.

The repeated evolution of herbicide resistance in weeds is an ongoing problem in agricultural regions across the world, and presents a unique system in which to study the origins and spread of adaptive traits across heterogeneous landscapes. Lolium perenne ssp. multiflorum (Lam.) (Italian ryegrass) is a widespread grass weed of agricultural crops that has repeatedly evolved resistance to herbicides across the world. In California, resistance to glyphosate has become increasingly common. To identify the mechanisms conferring glyphosate resistance in California populations of L. perenne and to gain insights into the evolutionary origins and spread of resistance in the region, we investigated the frequency of target-site mutations conferring resistance to glyphosate combined with the frequency of resistant individuals in 14 populations. A region of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was sequenced in 401 individuals to assay for target site mutations. Seven unique alleles were detected at codon site 106, four of which have been previously shown to confer target-site-based resistance to glyphosate. Four different resistance alleles were detected, indicating that resistance to glyphosate has evolved multiple times in the region. In two populations, no EPSPS mutations were detected despite the presence of resistant plants, strongly suggesting that non-target-site-based mechanisms confer resistance to glyphosate in these populations. It is likely that resistance to glyphosate in these 14 California populations of L. perenne derives from at least five evolutionary origins, indicating that adaptive traits can evolve repeatedly over agricultural landscapes.

Genetic diversity and structure of Lolium perenne ssp. multiflorum in California vineyards and orchards indicate potential for spread of herbicide resistance via gene flow.

Management of agroecosystems with herbicides imposes strong selection pressures on weedy plants leading to the evolution of resistance against those herbicides. Resistance to glyphosate in populations of Lolium perenne L. ssp. multiflorum is increasingly common in California, USA, causing economic losses and the loss of effective management tools. To gain insights into the recent evolution of glyphosate resistance in L. perenne in perennial cropping systems of northwest California and to inform management, we investigated the frequency of glyphosate resistance and the genetic diversity and structure of 14 populations. The sampled populations contained frequencies of resistant plants ranging from 10% to 89%. Analyses of neutral genetic variation using microsatellite markers indicated very high genetic diversity within all populations regardless of resistance frequency. Genetic variation was distributed predominantly among individuals within populations rather than among populations or sampled counties, as would be expected for a wide-ranging outcrossing weed species. Bayesian clustering analysis provided evidence of population structuring with extensive admixture between two genetic clusters or gene pools. High genetic diversity and admixture, and low differentiation between populations, strongly suggest the potential for spread of resistance through gene flow and the need for management that limits seed and pollen dispersal in L. perenne.

Molecular evidence for hybridization in Colias (Lepidoptera: Pieridae): are Colias hybrids really hybrids?

Gene flow and hybridization among species dramatically affect our understanding of the species as a biological unit, species relationships, and species adaptations. In North American Colias eurytheme and Colias eriphyle, there has been historical debate over the extent of hybridization occurring and the identity of phenotypically intermediate individuals as genetic hybrids. This study assesses the population structure of these two species to measure the extent of hybridization and the genetic identity of phenotypic intermediates as hybrids. Amplified fragment length polymorphism (AFLP) marker analysis was performed on 378 specimens collected from northern California and Nevada. Population structure was inferred using a Bayesian/Markov chain Monte Carlo method, which probabilistically assigns individuals to genetic clusters. Three genetic clusters provided the best fit for the data. C. eurytheme individuals were primarily assigned to two closely related clusters, and C. eriphyle individuals were mostly assigned to a third, more distantly related cluster. There appeared to be significant hybridization between the two species. Individuals of intermediate phenotype (putative hybrids) were found to be genetically indistinguishable from C. eriphyle, indicating that previous work based on the assumption that these intermediate forms are hybrids may warrant reconsideration.

Evolution and spread of glyphosate resistance in Conyza canadensis in California.

Recent increases in glyphosate use in perennial crops of California, USA, are hypothesized to have led to an increase in selection and evolution of resistance to the herbicide in Conyza canadensis populations. To gain insight into the evolutionary origins and spread of resistance and to inform glyphosate resistance management strategies, we investigated the geographical distribution of glyphosate resistance in C. canadensis across and surrounding the Central Valley, its spatial relationship to groundwater protection areas (GWPA), and the genetic diversity and population structure and history using microsatellite markers. Frequencies of resistant individuals in 42 sampled populations were positively correlated with the size of GWPA within counties. Analyses of population genetic structure also supported spread of resistance in these areas. Bayesian clustering and approximate Bayesian computation (ABC) analyses revealed multiple independent origins of resistance within the Central Valley. Based on parameter estimation in the ABC analyses, resistant genotypes underwent expansion after glyphosate use began in agriculture, but many years before it was detected. Thus, diversity in weed control practices prior to herbicide regulation in GWPA probably kept resistance frequencies low. Regionally coordinated efforts to reduce seed dispersal and selection pressure are needed to manage glyphosate resistance in C. canadensis.

POLYSAT: an R package for polyploid microsatellite analysis.

We present an R package to help remedy the lack of software for manipulating and analysing autopolyploid and allopolyploid microsatellite data. POLYSAT can handle genotype data of any ploidy, including populations of mixed ploidy, and assumes that allele copy number is always ambiguous in partial heterozygotes. It can import and export genotype data in eight different formats, calculate pairwise distances between individuals using a stepwise mutation and infinite alleles model, estimate ploidy based on allele counts and estimate allele frequencies and pairwise F(ST) values. This software is freely available through the Comprehensive R Archive Network (http://cran.r-project.org/) and includes a thorough tutorial.

Cross-resistance to herbicides of five ALS-inhibiting groups and sequencing of the ALS gene in Cyperus difformis L.

Resistance to ALS-inhibiting herbicides in Cyperus difformis has evolved rapidly in many rice areas worldwide. This study identified the mechanism of resistance, assessed cross-resistance patterns to all five chemical groups of ALS-inhibiting herbicides in four C. difformis biotypes, and attempted to sequence the ALS gene. Whole-plant and ALS enzyme activity dose-response assays indicated that the WA biotype was resistant to all ALS-inhibiting herbicides evaluated. The IR biotype was resistant to bensulfuron-methyl, orthosulfamuron, imazethapyr, and propoxycarbazone-sodium and less resistant to bispyribac-sodium and halosulfuron-methyl, and susceptible to penoxsulam. ALS enzyme activity assays indicated that resistance is due to an altered target site yet mutations previously found to endow target-site resistance in weeds were not detected in the sequences obtained. The inability to detect resistance mutations in C. difformis may result from the presence of additional ALS genes, which were not amplified by the primers used. This study reports the first ALS gene sequence from Cyperus difformis. Certain ALS-inhibiting herbicides can still be used to control some resistant C. difformis biotypes. However, because cross-resistance to all five classes of ALS-inhibitors was detected in other resistant biotypes, these herbicides should only be used within an integrated weed management program designed to delay the evolution of herbicide resistance.

Microsatellite variation points to local landscape plantings as sources of invasive pampas grass (Cortaderia selloana) in California.

International trade in horticultural plants is a major pathway of introduction of invasive species. Pampas grass (Cortaderia selloana) is an invasive species of horticultural origin that is native to South America but cultivated as an ornamental in regions with Mediterranean climates worldwide. To gain insight into the introduction history of invasive populations in California, we analysed microsatellite marker variation in cultivated and invasive C. selloana. We sampled 275 cultivated plants from diverse sources and 698 invasive plants from 33 populations in four geographical regions of California. A model-based Bayesian clustering analysis identified seven distinct gene pools in cultivated C. selloana. Probabilities of assignment of invasive individuals to cultivated gene pools indicated that two gene pools accounted for the genomic origin of 78% of the invasive C. selloana sampled. Extensive admixture between cultivated source gene pools was detected within invasive individuals. Sources of admixed invasive individuals are probably landscape plantings. Consistent with the Bayesian assignment results indicating that multiple cultivated gene pools and landscape plantings are probable sources of invasive populations, F(ST) and neighbour-joining clustering analyses indicated multiple escapes from shared sources in each geographical region. No isolation by distance or geographical trend in reduction of genetic diversity was evident. Furthermore, a generally random and discontinuous distribution of proportional assignments of invasive populations to cultivated gene pools suggests that introductions occurred recurrently within each geographical region. Our results strongly suggest that dispersal through local landscape plantings has contributed to the range expansion of invasive C. selloana in California.