Genomic Treasure Troves: Complete Genome Sequencing of Herbarium and Insect Museum Specimens

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today''s next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.     

Availability of short microsatellite markers from butterfly museums and private specimens

Knowledge of the temporal changes in genetic diversity and structure is important for identifying factors causing a decline in threatened insect species, and for establishing conservation programs for these species. Thus, there is recently an increasing interest in the restoration of genetic diversity in conservation programs using DNA data from historical museum specimens. For butterfly specimens, we measured the yields and fragment sizes of the extracted DNA and investigated the genotyping success probability of nine short microsatellite markers (allele size 73–191 bp). We used leg samples of specimens of a medium‐sized butterfly species, Melitaea ambigua (Lepidoptera; Nymphalidae), collected from the 1960s to the 2010s. The yields of specimen‐extracted DNA longer than 150 bp decreased with increasing specimen age. There were negative correlations between the genotyping success probability and specimen age for each of all microsatellite markers. A negative correlation was also observed between the genotyping success probability and allele size of each microsatellite marker. We conclude that short microsatellite markers and analysis of recently obtained specimens are particularly suitable for microsatellite analysis of butterfly specimens.     

How old can we go? Evaluating the age limit for effective DNA recovery from historical insect specimens

Historical museum specimens are valuable for exploring population genetics and evolutionary questions because they can provide snapshots of morphological and genetic characteristics from populations over space and time. Unfortunately, DNA found in older museum specimens is frequently degraded, so obtaining genotypes from many individual samples necessary for rigorous molecular population genetic studies is challenging. Previous studies have varied greatly in their success at obtaining genotypes from older preserved insect material. Many well-intentioned collection curators have used research results showing poor preservation of DNA preserved in museum specimens to inform curatorial best practices, in some cases choosing not to allow DNA extraction by destructive sampling because, in their estimation, the likelihood of success would be low. Recent methodological advances in DNA extraction, amplification, and genotyping have allowed some researchers to include mid-19th century samples in molecular genetic analyses. Here we present a robust, high-throughput, and low-cost DNA extraction and genotyping protocol for historical insect specimens employing restriction digests of PCR products followed by high sensitivity electrophoresis. Using this technique, we obtained mitochondrial haplotypes for 100% of 48 New World Junonia butterfly specimens (Nymphalidae) ranging in age from pre-1813 to 1909 and show that the haplotype frequencies obtained are statistically indistinguishable from 20th-century and contemporary reference populations of Junonia (1632 specimens) matched by geographic region. As most extant insect specimens were collected after 1813, based on our findings we would expect that many or even most pinned specimens preserved in museum collections contain usable DNA for mitochondrial haplotyping.     

Museomics Dissects the Genetic Basis for Adaptive Seasonal Coloration in the Least Weasel

Dissecting the link between genetic variation and adaptive phenotypes provides outstanding opportunities to understand fundamental evolutionary processes. Here, we use a museomics approach to investigate the genetic basis and evolution of winter coat coloration morphs in least weasels (Mustela nivalis), a repeated adaptation for camouflage in mammals with seasonal pelage color moults across regions with varying winter snow. Whole-genome sequence data were obtained from biological collections and mapped onto a newly assembled reference genome for the species. Sampling represented two replicate transition zones between nivalis and vulgaris coloration morphs in Europe, which typically develop white or brown winter coats, respectively. Population analyses showed that the morph distribution across transition zones is not a by-product of historical structure. Association scans linked a 200-kb genomic region to coloration morph, which was validated by genotyping museum specimens from intermorph experimental crosses. Genotyping the wild populations narrowed down the association to pigmentation gene MC1R and pinpointed a candidate amino acid change cosegregating with coloration morph. This polymorphism replaces an ancestral leucine residue by lysine at the start of the first extracellular loop of the protein in the vulgaris morph. A selective sweep signature overlapped the association region in vulgaris, suggesting that past adaptation favored winter-brown morphs and can anchor future adaptive responses to decreasing winter snow. Using biological collections as valuable resources to study natural adaptations, our study showed a new evolutionary route generating winter color variation in mammals and that seasonal camouflage can be modulated by changes at single key genes.     

Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.     

Historical and non‐invasive samples: a study case of genotyping errors in newly isolated microsatellites for the lesser anteater (Tamandua tetradactyla L., Pilosa)

Tamandua tetradactyla (Pilosa), the lesser anteater, is a medium‐size mammal from South America. Its wide distribution through different landscapes, solitary and nocturnal habits, and the difficulty to capture and contain specimens limit the amount of individuals and populations sampled during fieldworks. These features along with the lack of specific molecular markers for the lesser anteater might be the causes for paucity in population genetic studies for the species. Historical samples from museum specimens, such as skins, and non‐invasive samples, such as plucked hair, can be supplementary sources of DNA samples. However, the DNA quantity and quality of these samples may be limiting factors in molecular studies. In this study, we describe nine microsatellite loci for T. tetradactyla and test the amplification success, data reliability and estimate errors on both historical and non‐invasive sample sets. We tested nine polymorphic microsatellites and applied the quality index approach to evaluate the relative performance in genotype analysis of 138 historical samples (study skin) and 19 non‐invasive samples (plucked hair). The observed results show a much superior DNA quality of non‐invasive over historical samples and support the quality index analysis as a practical tool to exclude samples with doubtful performance in genetic studies. We also found a relationship between the age of non‐invasive samples and DNA quality, but lack of evidence of this pattern for historical samples.     

DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.     

Singing from the Grave: DNA from a 180 Year Old Type Specimen Confirms the Identity of Chrysoperla carnea (Stephens)

Historically serving as repositories for morphologically-based taxonomic research, natural history collections are now increasingly being targeted in studies utilizing DNA data. The development of advanced molecular techniques has facilitated extraction of useable DNA from old specimens, including type material. Sequencing diagnostic molecular markers from type material enables accurate species designation, especially where modern taxonomic hypotheses confirm morphologically cryptic species complexes. One such example is Chrysoperla carnea (Stephens), which belongs to a complex of about 20 cryptic species, most of which can only be reliably distinguished by their pre-mating courtship songs or by DNA analysis. The subtle morphological variation in the group has led to disagreement over the previous designation of the lectotype for C. carnea, an issue that has been further compounded because Chrysoperla carnea is a highly valued biological control agent in arable crops. Archival DNA extraction and sequencing from the 180 year old lectotype specimen, combined with Bayesian and Likelihood based phylogenetic analyses of modern specimens from the entire complex, were used to establish unambiguously the true identity of Chrysoperla carnea.     

Unearthing the impact of human disturbance on a notorious weed

Large‐scale anthropogenic changes in the environment are reshaping global biodiversity and the evolutionary trajectory of many species. Evolutionary mechanisms that allow organisms to thrive in this rapidly changing environment are just beginning to be investigated (Hoffmann & Sgrò 2011 ; Colautti & Barrett 2013 ). Weedy and invasive species represent ‘success stories’ for how species can cope with human modified environments. As introduced species have spread within recent times, they provide the unique opportunity to track the genetic consequences of rapid range expansion through time and space using historic DNA samples. Using modern collections and herbarium specimens dating back to 1873, Martin et al. ( 2014 ) have provided a more complete understanding of the population history of the invasive, agricultural weed, common ragweed (Ambrosia artemisiifolia; Fig.  1 ) in its native range with surprising results. They find that the recent population explosion of common ragweed in North America coincided with substantial shifts in population genetic structure with implications for invasion.     

A comprehensive DNA barcode library for the looper moths (Lepidoptera: Geometridae) of British Columbia, Canada

Background

The construction of comprehensive reference libraries is essential to foster the development of DNA barcoding as a tool for monitoring biodiversity and detecting invasive species. The looper moths of British Columbia (BC), Canada present a challenging case for species discrimination via DNA barcoding due to their considerable diversity and limited taxonomic maturity.

Methodology/Principal Findings

By analyzing specimens held in national and regional natural history collections, we assemble barcode records from representatives of 400 species from BC and surrounding provinces, territories and states. Sequence variation in the barcode region unambiguously discriminates over 93% of these 400 geometrid species. However, a final estimate of resolution success awaits detailed taxonomic analysis of 48 species where patterns of barcode variation suggest cases of cryptic species, unrecognized synonymy as well as young species.

Conclusions/Significance

A catalog of these taxa meriting further taxonomic investigation is presented as well as the supplemental information needed to facilitate these investigations.     

Evidence of a Strong Domestication Bottleneck in the Recently Cultivated New Zealand Endemic Root Crop,Arthropodium cirratum (Asparagaceae)

We use chloroplast DNA sequencing to examine aspects of the pre-European Māori cultivation of an endemic New Zealand root crop, Arthropodium cirratum (rengarenga). Researching the early stages of domestication is not possible for the majority of crops, because their cultivation began many thousands of years ago and/or they have been substantially altered by modern breeding methods. We found high levels of genetic variation and structuring characterised the natural distribution of A. cirratum, while the translocated populations only retained low levels of this diversity, indicating a strong bottleneck even at the early stages of this species’ cultivation. The high structuring detected at four chloroplast loci within the natural A. cirratum range enabled the putative source(s) of the translocated populations to be identified as most likely located in the eastern Bay of Plenty/East Cape region. The high structuring within A. cirratum also has implications for the conservation of genetic diversity within this species, which has undergone recent declines in both its natural and translocated ranges.     

A study of the genetic variation of the aquatic fern Marsilea quadrifolia L. preserved in botanical collections in Poland and originated from natural populations in Europe

Aim of the present study was to evaluate the genetic diversity of selected European populations of Marsilea quadrifolia L. and to assess the applicability of those genetic resources of Marsilea quadrifolia L. that have been preserved in Polish botanical gardens, for the reintroduction of this species into its historical range in Poland. Three Polish populations that originated from botanical collections (Zabrze, ?arów and Pu?awy) and four natural populations (two from Slovakia (Slovakia I and Slovakia II), one from France and one from Germany) were analyzed using Amplified Fragment Length Polymorphism (AFLP) markers. A very low level of genetic variation was found both within and between the populations in the study, which likely resulted from a genetic bottleneck probably caused by human activities. Plants with the same AFLP fingerprint were found across several populations; however, singleton samples with a unique AFLP band pattern were also present within all of the populations. The presence of singletons led to relatively high values of Simpson's diversity index, which may suggest a considerable effect of mutations and some possibility of sexual reproduction as sources of the observed variation. The partitioning of molecular variance was calculated using hierarchical AMOVA, which showed that a negligible value of only 0.81% of the variation was explained by the category of population, i.e. plants originating from the botanical collections or from the natural habitats. This result indicates that M. quadrifolia populations from botanical collections resemble natural populations in terms of the level of their genetic variation and that the populations that were obtained from the Polish collections could be used for the successful reintroduction of this species into its historical range in Poland, and a similar situation may be given also in other areas of occurrence of this plant that is under threat throughout its area of occurrence in Europe.     

Can genetic data confirm or refute historical records? The island invasion of the small Indian mongoose (Herpestes auropunctatus)

Many studies aimed at reconstructing the invasion history of a species rely, in part, on inferences based on patterns of genetic variation. These inferences warrant careful interpretation, however. In particular, given the time scale of most invasions, the typical demography of invasive species in their invaded range, and the available molecular tools, the underlying assumptions of population genetic models will often be violated. Given this fact, we examined the potential of population genetic data for reconstructing the history of serial introductions of the small Indian mongoose, Herpestes auropunctatus. We used simulations to test the power of existing microsatellite data for testing the credibility of historical introduction records. Although our results are generally consistent with most historical records for H. auropunctatus, the existing data have low power to reject alternative historical hypotheses. Simulations of a wide range of founder population sizes show broadly overlapping results, making rather different historical scenarios of introductions difficult to rule out with typical datasets. We advocate caution in the use of molecular population genetics to infer the history of invasive species, and we suggest extensive simulations as a tool to evaluate, in advance, this approach for addressing important research questions.     

Back to the future: museum specimens in population genetics

Museums and other natural history collections (NHC) worldwide house millions of specimens. With the advent of molecular genetic approaches these collections have become the source of many fascinating population studies in conservation genetics that contrast historical with present-day genetic diversity. Recent developments in molecular genetics and genomics and the associated statistical tools have opened up the further possibility of studying evolutionary change directly. As we discuss here, we believe that NHC specimens provide a largely underutilized resource for such investigations. However, because DNA extracted from NHC samples is degraded, analyses of such samples are technically demanding and many potential pitfalls exist. Thus, we propose a set of guidelines that outline the steps necessary to begin genetic investigations using specimens from NHC.     

Museum epigenomics: Characterizing cytosine methylation in historic museum specimens

Museum genomics has transformed the field of collections‐based research, opening up a range of new research directions for paleontological specimens as well as natural history specimens collected over the past few centuries. Recent work demonstrates that it is possible to characterize epigenetic markers such as DNA methylation in well preserved ancient tissues. This approach has not yet been tested in traditionally prepared natural history specimens such as dried bones and skins, the most common specimen types in vertebrate collections. In this study, we developed and tested methods to characterize cytosine methylation in dried skulls up to 76 years old. Using a combination of ddRAD and bisulphite treatment, we characterized patterns of cytosine methylation in two species of deer mouse (Peromyscus spp.) collected in the same region in Michigan in 1940, 2003, and 2013–2016. We successfully estimated methylation in specimens of all age groups, although older specimens yielded less data and showed greater interindividual variation in data yield than newer specimens. Global methylation estimates were reduced in the oldest specimens (76 years old) relative to the newest specimens (1–3 years old), which may reflect post‐mortem hydrolytic deamination. Methylation was reduced in promoter regions relative to gene bodies and showed greater bimodality in autosomes relative to female X chromosomes, consistent with expectations for methylation in mammalian somatic cells. Our work demonstrates the utility of historic specimens for methylation analyses, as with genomic analyses; however, studies will need to accommodate the large variance in the quantity of data produced by older specimens.     

Long-term population genetic structure of an invasive urochordate: the ascidian Botryllus schlosseri

The accelerated pace of marine biological invasions raises questions pertaining to genetic traits and dynamics underlying the successful establishment of invasive species. Current research stresses the importance of multiple introductions and prolonged gene flow as the main sources for genetic diversity, which, along with genetic drift, affect invasive species success. We here attempt to determine the relative contribution of gene flow and mutation rates as sources of genetic variability using the invasive tunicate Botryllus schlosseri as a model. The study was performed over a 13-year period in the Santa Cruz Harbor, California. With a characteristic life history of five generations/year, the Santa Cruz Botryllus population has already experienced approximately 155 generations since the onset of its invasion. The results (278 specimens, 127 scored alleles, five microsatellite loci) support limited gene flow rate (2.89?×?10^?3) and relative genetic isolation. Furthermore, the study population was found to be influenced by both, genetic drift and a high mutation rate (2.47?×?10^?2). These findings were supported by high fluctuations in the frequencies of microsatellite alleles, the appearance of new alleles and the loss of others. The balance between genetic drift and a high mutation rate is further elucidated by the high, stable level of genetic variation. We suggest that rapid mutation rates at the microsatellite loci reflect genome-wide phenomena, helping to maintain high genetic variability in relatively isolated populations. The potential adaptability to new environments is discussed.     

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence

Background

The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum.

Results

Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, inter-population differentiation, and the degree to which allele frequencies are correlated between populations.

Conclusions

The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.     

Sequence capture phylogenomics of historical ethanol‐preserved museum specimens: Unlocking the rest of the vault

Natural history collections play a crucial role in biodiversity research, and museum specimens are increasingly being incorporated into modern genetics‐based studies. Sequence capture methods have proven incredibly useful for phylogenomics, providing the additional ability to sequence historical museum specimens with highly degraded DNA, which until recently have been deemed less valuable for genetic work. The successful sequencing of ultraconserved elements (UCEs) from historical museum specimens has been demonstrated on multiple tissue types including dried bird skins, formalin‐fixed squamates and pinned insects. However, no study has thoroughly demonstrated this approach for historical ethanol‐preserved museum specimens. Alongside sequencing of “fresh” specimens preserved in >95% ethanol and stored at ?80°C, we used extraction techniques specifically designed for degraded DNA coupled with sequence capture protocols to sequence UCEs from historical museum specimens preserved in 70%–80% ethanol and stored at room temperature, the standard for such ethanol‐preserved museum collections. Across 35 fresh and 15 historical museum samples of the arachnid order Opiliones, an average of 345 UCE loci were included in phylogenomic matrices, with museum samples ranging from six to 495 loci. We successfully demonstrate the inclusion of historical ethanol‐preserved museum specimens in modern sequence capture phylogenomic studies, show a high frequency of variant bases at the species and population levels, and from off‐target reads successfully recover multiple loci traditionally sequenced in multilocus studies including mitochondrial loci and nuclear rRNA loci. The methods detailed in this study will allow researchers to potentially acquire genetic data from millions of ethanol‐preserved museum specimens held in collections worldwide.     

Genetic Bottlenecks in Time and Space: Reconstructing Invasions from Contemporary and Historical Collections

Herbarium accession data offer a useful historical botanical perspective and have been used to track the spread of plant invasions through time and space. Nevertheless, few studies have utilised this resource for genetic analysis to reconstruct a more complete picture of historical invasion dynamics, including the occurrence of separate introduction events. In this study, we combined nuclear and chloroplast microsatellite analyses of contemporary and historical collections of Senecio madagascariensis, a globally invasive weed first introduced to Australia c. 1918 from its native South Africa. Analysis of nuclear microsatellites, together with temporal spread data and simulations of herbarium voucher sampling, revealed distinct introductions to south-eastern Australia and mid-eastern Australia. Genetic diversity of the south-eastern invasive population was lower than in the native range, but higher than in the mid-eastern invasion. In the invasive range, despite its low resolution, our chloroplast microsatellite data revealed the occurrence of new haplotypes over time, probably as the result of subsequent introduction(s) to Australia from the native range during the latter half of the 20^th century. Our work demonstrates how molecular studies of contemporary and historical field collections can be combined to reconstruct a more complete picture of the invasion history of introduced taxa. Further, our study indicates that a survey of contemporary samples only (as undertaken for the majority of invasive species studies) would be insufficient to identify potential source populations and occurrence of multiple introductions.     

Herbarium specimens reveal a historical shift in phylogeographic structure of common ragweed during native range disturbance

Invasive plants provide ample opportunity to study evolutionary shifts that occur after introduction to novel environments. However, although genetic characters pre‐dating introduction can be important determinants of later success, large‐scale investigations of historical genetic structure have not been feasible. Common ragweed (Ambrosia artemisiifolia L.) is an invasive weed native to North America that is known for its allergenic pollen. Palynological records from sediment cores indicate that this species was uncommon before European colonization of North America, and ragweed populations expanded rapidly as settlers deforested the landscape on a massive scale, later becoming an aggressive invasive with populations established globally. Towards a direct comparison of genetic structure now and during intense anthropogenic disturbance of the late 19th century, we sampled 45 natural populations of common ragweed across its native range as well as historical herbarium specimens collected up to 140 years ago. Bayesian clustering analyses of 453 modern and 473 historical samples genotyped at three chloroplast spacer regions and six nuclear microsatellite loci reveal that historical ragweed's spatial genetic structure mirrors both the palaeo‐record of Ambrosia pollen deposition and the historical pattern of agricultural density across the landscape. Furthermore, for unknown reasons, this spatial genetic pattern has changed substantially in the intervening years. Following on previous work relating morphology and genetic expression between plants collected from eastern North America and Western Europe, we speculate that the cluster associated with humans’ rapid transformation of the landscape is a likely source of these aggressive invasive populations.