Biology subjects

Thompson, K. A.

Publications and source records attributed to Thompson, K. A..

At least 19 recordsLinked to original sources

bioRxiv2026

The Barcode Inference Pipeline (BIP): From Sequencer Output to DNA Barcodes

DNA barcoding involves the recovery of a DNA sequence for a target gene region from its source specimen. This process gains complexity when multiple sequences are recovered from a specimen, as is often the case when data are generated by high-throughput sequencers. This diversity can reflect both methodological artifacts (e.g., chimeras, PCR errors, sequencing errors, tag jumps) and real template diversity in the DNA extract (e.g., contamination, endosymbionts, NUMTs, parasites). To support analysis of the sequence data from three million specimens annually, the Centre for Biodiversity Genomics (CBG) has developed BIP, the Barcode Inference Pipeline. Compatible with all sequencing platforms, BIP processes .fastq files and returns both target DNA barcodes and non-target sequences. To generate results, BIP implements quality and size filtration, demultiplexing, primer trimming, chimera scanning, sequence error correction, OTU delineation, and sequence identification. When analysis targets the cytochrome c oxidase 1 (COI) barcode region, BIP also assigns each OTU to a known BIN or identifies its nearest neighbour BIN. As final output, BIP returns summary files ready for upload to BOLD or for other downstream analyses. They include a taxonomic assignment for each OTU, generated by comparison with a DNA barcode reference library. We describe BIPs flexibility and structure, then demonstrate its functionality by analyzing COI sequence data from 100K specimens. Because of its capacity to disentangle target and non-target sequences, BIP outperforms an alternative software package, ONTbarcoder, in several important ways. To ease access, installation, and functionality, BIP is provided as a Docker container (github.com/cbg-innov/BIP).

bioinformatics↗

bioRxiv2026

The Metabarcoding Analysis Pipeline (MAP): Simple, accurate, and flexible metabarcoding

Current metabarcoding pipelines are inflexible with respect to study design and are poorly suited to long-read sequence data. To address these limitations, we developed MAP, the Metabarcoding Analysis Pipeline, which is a sequence-to-answer workflow supporting the analysis of amplicons from highly multiplexed and replicated study designs. Although MAP can analyze amplicons of any length from any genetic marker, it includes several features tailored to long-read COI metabarcoding. MAP installs from a Docker container and requires only sequence data, a parameters file, and a reference library. It produces intuitive reports, enabling users to evaluate their data immediately after analysis. We validate MAP by showing that it generates biodiversity estimates that correspond closely to a ground-truth dataset of single-specimen DNA barcode data and by demonstrating that it outperforms alternative platforms for COI metabarcoding. MAP is free, open-source, and available from: https://github.com/cbg-innov/MAP.

bioinformatics↗

bioRxiv2026

Spatial Variability in Soil Necrobiome Communities has a Negligible Effect on Postmortem Interval Estimation

Microbial succession can be used to estimate the postmortem interval (PMI); however, the impact of spatial variability within the cadaver decomposition island (CDI) is not well understood. This study examined spatial variation in necrobiome communities where soil samples were collected over time and across spatial locations from the CDIs of two human body donors. Microbial communities were characterized using 16S rRNA sequencing and statistical modelling of variation and PMI were conducted. Necrobiome community metrics showed no significant differences across anatomical sampling sites within the CDI at a single timepoint. Temporal modelling identified 11 taxa with significant relationships to PMI in one donor, with spatial sampling having a minimal impact on the PMI relationships. Non-linear approaches also identified taxa with likely PMI signals in the second donor. These findings demonstrate that opportunistic sampling can capture robust linear and non-linear PMI signals in later decomposition stages.

microbiology↗

bioRxiv2026

Performance Test of the QNome Nanopore Sequencer

Nanopore sequencers have the potential to liberate DNA sequencing from centralized core facilities to distributed analytical nodes. Until now, Oxford Nanopore Technologies (ONT) has been the sole manufacturer of a portable nanopore sequencer, but analogous platforms are in production. Nanopore sequencers from Qitan Technology (QT) are widely used in China but have been unavailable outside that nation and lack independent performance testing. Enabled by early access to QTs least expensive sequencer and flow cell, the QNome-3841 and QCell-384, we tested whether they could generate accurate DNA barcodes cost-effectively. In several tests involving amplicon pools from 95 to 9,120 specimens, QT recovered valid DNA barcodes from nearly as many specimens (98%) as ONT. QT sequences had slightly lower fidelity than their ONT counterparts and QT frequently failed to resolve the correct length of G/C homopolymers. However, barcode sequences from the two platforms were nearly indistinguishable after bioinformatic treatment. QTs wash kit performed well, enabling a QCell to sequence eight amplicon pools with zero carryover between runs and minimal degradation of the flow cell. Its ultra-fast protocol allowed library preparation in a single step that could be completed in 15 minutes, but this came at the cost of lower quality data. Once widely available, QT devices will be well-suited for supporting DNA barcode analysis.

genomics↗

bioRxiv2024

The historical biogeography of range expansion predicts spatial patterns in reproductive assurance

Plant reproductive assurance describes the ability of a plant to successfully reproduce in an environment that is potentially devoid of conspecifics and/or pollinators. Traditionally, studies have focused on the role of contemporary ecology---such as pollinator or mate availability---in driving spatial patterns in reproductive assurance within species, however, historical processes such as post-glacial range expansion may be an understudied alternative explanation for geographic variation in mating system. This is because during the process of geographic range expansion into novel habitat, selection should favour individuals that possess traits promoting reproductive assurance (i.e., autonomous selfing or clonal reproduction). Here, we used Northern pink monkeyflower--Erythranthe (Mimulus lewisii--a hermaphroditic, self-compatible, perennial, alpine plant, as a focal species to investigate the historical signatures of geographic range expansion by combining phylogeographic analyses with a greenhouse survey of range-wide reproductive assurance. First, we detected significant geographic variation among populations in two components of reproductive assurance: self-fertilization and clonal propagation. Next, using genome-wide single nucleotide polymorphism (SNP) data, we identified three distinct genetic clusters structured by longitude and estimated geographic coordinates of the most likely origins of range expansion within each cluster. We found that both measures of reproductive assurance significantly increased on average with distance from the inferred biogeographic origin, which is consistent with the hypothesis that reproductive assurance undergoes adaptive evolution during range expansion. This study supports hypotheses underlying Bakers Law and contributes to our understanding of spatial variation in reproductive assurance, which is linked to variation in evolutionary potential, adaptability, and the long-term persistence of populations across large spatial scales. Lay summaryWhat generates and maintains the amazing diversity in plant reproduction we see today? Traditionally, studies have focused on the role of contemporary ecology, such as pollinator or mate availability, in driving spatial patterns within species, however, historical drivers may be an understudied alternative explanation. Here, we test the hypothesis that expansion into new habitats after glacial retreat, where pollinators or mates were scarce, favors plants with the ability to reproduce in the absence of pollinators and mates by making self-pollinated seeds or vegetative clones of themselves. We find support for the hypothesis, where the probabilities of making both autonomously selfed seed and clonal vegetative propagules increase on average along inferred pathways of range expansion. Our study joins a handful of previous studies that tested and provided support for historical range expansion shaping range-wide spatial variation in reproductive assurance in plants, suggesting post-glacial range expansion may be a common mechanism creating diversity in plant mating systems.

evolutionary biology↗

bioRxiv2023

Evidence for kilometer-scale biophysical features at the Gulf Stream front

Understanding the interplay of ocean physics and biology, particularly at the submesoscale and below (<30km), is an ongoing challenge in oceanography. While poorly constrained, these scales may be of critical importance for understanding how changing ocean dynamics will impact marine ecosystems. Fronts in the ocean, regions where two disparate water masses meet and isopycnals become tilted towards vertical, are considered hotspots for biophysical interaction, but there is limited observational evidence at the appropriate scales to assess their importance. Western boundary currents like the Gulf Stream are of particular interest as these dynamic physical regions are thought to influence both productivity and composition of primary producers; however, how exactly this plays out, and at what scales, is not well known. Using satellite data and two years of detailed in situ observations across the Gulf Stream front near Cape Hatteras, North Carolina, U.S.A., we investigate how submesoscale frontal dynamics could affect biological communities associated with frontal regions and generate hotspots of productivity and export. In this analysis we assess the seasonality and phenology of the region, generalize the kilometer-scale structure of the front, and analyze 69 transects to assess two physical processes of potential biogeochemical importance: cold shelf filament subduction and high salinity Sargasso Sea obduction. We link these processes observationally to the meander phase of the Gulf Stream and discuss how cold filament subduction could be exporting carbon and how obduction of high salinity water from depth often leads to high chlorophyll-a. Finally, we report on phytoplankton community composition in each of these features and integrate these new observations into our understanding of frontal submesoscale dynamics. Plain Language SummaryPhytoplankton move with large currents and are stirred by eddies with diameters ranging from 100s of kilometers down to the meter scale. Their growth is impacted by physical factors like light and temperature and also chemical and biological factors like nutrient availability, and their accumulation is also impacted by top down controls (zooplankton grazing, viral lysis) and competition with other phytoplankton. This interplay of physics and biology in determining the biomass and composition of phytoplankton communities is poorly understood and is key to understanding marine ecosystem resilience and structure in a changing ocean. In this work we investigated the impact of physics and biology on phytoplankton across scales focusing on the Gulf Stream front. Fronts in the ocean are where lines of equal density go from being horizontal to having a vertical tile, and because of this can enable nutrients and plankton to move from depth to the surface and vice versa. The objective of this work is to understand how physics might drive important changes in phytoplankton biomass and composition in the Gulf Stream front, which is amongst the sharpest gradients in temperature, density, and current speed in the global ocean. We find two frequent processes at the front, the apparent subduction of cold filaments down along the edge of the Gulf Stream, associated with meander troughs, and obduction of high salinity Sargasso Sea water into the front linked to meander crests. While ephemeral, these processes are frequent and could have a large impact on local phytoplankton biomass, phytoplankton composition, and the export of organic matter to depth. Key PointsO_LIThe frontal zone between the Gulf Stream and the shelf has an interface water mass which appears to have different origins with a range of biogeochemical impacts. C_LIO_LIMeanders appear to largely control the frontal interface: troughs lead to subduction of shelf filaments and crests lead to obduction of high salinity water. C_LIO_LIThese two processes are common at the front and could lead to ephemeral kilometer- scale export and productivity. C_LI

ecology↗

bioRxiv2023

A positive association between gut microbiota diversity and vertebrate host performance in a field experiment

The vertebrate gut microbiota is a critical determinant of organismal function, yet it remains unclear if and how gut microbial communities affect host fitness under natural conditions. Here, we investigate associations between growth rate (a fitness proxy) and gut microbiota diversity and composition in a field experiment with threespine stickleback fish (Gasterosteus aculeatus). We detected on average 63% more bacterial taxa in the guts of high-fitness fish compared to low-fitness fish (i.e., higher -diversity), suggesting that higher diversity promotes host growth. The microbial communities of high-fitness fish had higher similarity (i.e., lower {beta}-diversity) than low-fitness fish, supporting the Anna Karenina principle-- that there are fewer ways to have a functional microbiota than a dysfunctional microbiota. Our findings provide a basis for functional tests of the fitness consequences of host-microbiota interactions. Significance statementThe vertebrate gut microbiota is important for many aspects of their hosts biology--such as nutrient metabolism and defense against pathogens--that could ultimately affect host fitness. However, studies investigating the effects of gut microbiota composition on vertebrate host fitness under natural conditions remain exceedingly rare. We tested for associations between gut microbiota diversity and growth rate (a fitness proxy) in threespine stickleback fish reared in large outdoor ponds. We found evidence that a more diverse gut microbiota was predictive of higher growth rate and therefore increased host fitness. Notably, high-fitness fish had higher gut microbiota similarity to one another than did low-fitness fish, providing experimental evidence for the Anna Karenina principle--that there are fewer ways to have a functional microbiota than a dysfunctional microbiota--as it relates to host fitness.

evolutionary biology↗

bioRxiv2022

Heterosis counteracts hybrid breakdown to forestall speciation by parallel natural selection

In contrast to ecological speciation, where reproductive isolation evolves as a consequence of divergent natural selection, speciation by parallel natural selection has been less thoroughly studied. To test whether parallel evolution drives speciation, we leveraged the repeated evolution of benthic and limnetic ecotypes of threespine stickleback fish and estimated fitness for pure crosses and within-ecotype hybrids in semi-natural ponds and in laboratory aquaria. In ponds, we detected hybrid breakdown in both ecotypes but this was counterbalanced by heterosis and the strength of post-zygotic isolation was nil. In aquaria, we detected heterosis only in limnetic crosses and breakdown in neither ecotype, suggesting that hybrid incompatibilities are environment-dependent for both ecotypes and that heterosis is environment-dependent in benthic crosses. Heterosis and breakdown were 3x greater in limnetic crosses than in benthic crosses, contrasting the prediction that the fitness consequences of hybridization should be greater in crosses among more derived ecotypes. Consistent with a primary role for stochastic processes, patterns differed among crosses between populations from different lakes. Yet, we observed qualitatively similar patterns of heterosis and hybrid breakdown in benthic crosses and limnetic crosses when averaging the lake pairs, suggesting that the outcome of hybridization is repeatable in a general sense.

evolutionary biology↗

bioRxiv2021

START domain mediates Arabidopsis GLABRA2 transcription factor dimerization and turnover independently of homeodomain DNA binding

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding homeodomain (HD) in conjunction with a lipid-binding domain termed START (Steroidogenic Acute Regulatory (StAR)-related lipid Transfer). Previous work established that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis, is required for transcription factor activity. Here, we address the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) result in phenotypic defects in leaf trichomes, root hairs and seed mucilage, similar to those observed for START domain mutants, despite nuclear localization of the respective proteins. In vitro and in vivo experiments demonstrate that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, protein interaction assays reveal impaired GL2 dimerization for multiple alleles of START mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provide evidence for the role of START, but not HD, in maintaining protein stability. This work advances our mechanistic understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.

plant biology↗

bioRxiv2021

Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism

Plant homeodomain leucine-zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. The START domain is required for TF activity; however, its presumed role as a lipid sensor is not well understood. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative family member which controls epidermal differentiation, recruits lysophosphatidylcholines in a START-dependent manner. In vitro assays with recombinant protein verified that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally uncovered that PDF2 controls the expression of phospholipid-related target genes by binding to a palindromic octamer with consensus to a phosphate (Pi) response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments further revealed a role for START in maintaining protein levels, and Pi limitation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Overall our data provide insights towards understanding how the lipid metabolome integrates Pi availability with gene expression.

plant biology↗

bioRxiv2021

How dominant alleles influence progress towardspeciation by natural selection

When divergent populations interbreed, their alleles are brought together in hybrids. These hybrids may express novel phenotypes, not previously exposed to selection. In the initial F1 cross, most divergent alleles are present as heterozygotes. Therefore, F1 fitness can be influenced by dominance effects that first appear together in the hybrids, and so could not have been selected to function well together. We present a systematic study of these F1 dominance effects by introducing variable phenotypic dominance into Fishers geometric model. We show that dominance often reduces hybrid fitness, which can lead to patterns of optimal outbreeding and a steady decline in F1 fitness at high levels of divergence. We also show that "lucky" beneficial effects sometimes arise by chance, which might be especially important when hybrids can access novel environments. We then explore the interaction of phenotypic dominance with uniparental inheritance, showing that dominance can lead to violations of Haldanes Rule (reduced fitness of the heterogametic sex) while strengthening Darwins Corollary (fitness differences between cross directions). Taken together, our results show that dominance could play an important role in the outcomes of hybridisation after secondary contact, and thus to the maintenance or collapse of isolating barriers. Nevertheless, the telltale signs of dominance are relatively few and subtle. Results also suggest that dominance effects are smaller than the cost of segregation variance, implying that simple additive models may still give good predictions for later-generation recombinant hybrids, even when dominance qualitatively alters outcomes for the F1.

evolutionary biology↗

bioRxiv2021

Genetic evidence for environment-dependent hybrid incompatibilities in threespine stickleback

Hybrid incompatibilities occur when interactions between opposite-ancestry alleles at different loci reduce the fitness of hybrids. Most work on incompatibilities has focused on those that are intrinsic, meaning they affect viability and sterility in the laboratory. Theory predicts that ecological selection can also underlie hybrid incompatibilities, but tests of this hypothesis are scarce. In this article, we compiled genetic data for F2 hybrid crosses between divergent populations of threespine stickleback fish (Gasterosteus aculeatus L.) that were born and raised in either the field (semi-natural experimental ponds) or the laboratory (aquaria). We tested for differences in excess heterozygosity between these two environments at ancestry informative loci--a genetic signature of selection against incompatibilities. We found that excess ancestry heterozygosity was elevated by approximately 3% in crosses raised in ponds compared to those raised in aquaria. Previous results from F1 hybrids in the field suggest that pond-specific (single-locus) heterosis is unlikely to explain this finding. Our study suggests that, in stickleback, a coarse signal of environment-dependent hybrid incompatibilities is reliably detectable and that extrinsic incompatibilities have evolved before intrinsic incompatibilities.

evolutionary biology↗

bioRxiv2020

Adaptive divergence and the evolution of hybrid phenotypes in threespine stickleback

Hybrid fitness can be negatively impacted by trait mismatch, whereby hybrids resemble one parent population for some phenotypic traits and the other parent population for other traits. In this study, we used threespine stickleback fish (Gasterosteus aculeatus L.) to test whether trait mismatch in hybrids increases with the magnitude of phenotypic divergence between parent populations. We measured morphological traits in parents and hybrids in crosses between a marine population representing the ancestral form and twelve freshwater populations that have diverged from this ancestral state to varying degrees according to their environments. We found that trait mismatch was greater in more divergent crosses for both F1 and F2 hybrids. In the F1, the divergence-mismatch relationship was caused by traits having dominance in different directions whereas it was caused by segregating phenotypic variation in the F2. Selection against mismatched traits is an ecological analogue to selection against intrinsic hybrid incompatibilities, and our results imply that extrinsic hybrid incompatibilities accumulate predictably as phenotypic divergence proceeds.

evolutionary biology↗

bioRxiv2020

Climate warming weakens local adaptation

Anthropogenic climate change is generating mismatches between the environmental conditions that populations historically experienced and those in which they reside. Understanding how climate change affects population performance is a critical scientific challenge. We combine a quantitative synthesis of field transplant experiments with a novel statistical approach based in evolutionary theory to quantify the effects of temperature and precipitation variability on population performance. We find that species average performance is affected by both temperature and precipitation, but populations show signs of local adaptation to temperature only. Contemporary responses to temperature are strongly shaped by the local climates under which populations evolved, resulting in performance declines when temperatures deviate from historic conditions. Adaptation to other local environmental factors is strong, but temperature deviations as small as 2{degrees}C erode the advantage that these non-climatic adaptations historically gave populations in their home sites. One sentence summaryClimate change is pulling the thermal rug out from under populations, reducing average performance and eroding their historical home-site advantage.

evolutionary biology↗

bioRxiv2019

Patterns, predictors, and consequences of dominance in hybrids

Are first-generation (F1) hybrids typically intermediate for all traits that differentiate their parents? Or are they similar to one parent for most traits, or even mismatched for divergent traits? Although the phenotype of otherwise viable and fertile hybrids determines their fate, little is known about the general patterns, predictors, and consequences of phenotype expression in hybrids. To address this empirical gap, we compiled data from nearly 200 studies where traits were measured in a common environment for two parent populations and F1 hybrids. We find that individual traits are typically halfway between the parental midpoint and one parental value (i.e., hybrid trait values are typically 0.25 or 0.75 if parents values are 0 & 1). When considering pairs of traits together, a hybrids multivariate phenotype tends to resemble one parent (pairwise parent-bias) about 50 % more than the other while also exhibiting a similar magnitude of trait mismatch due to different traits having dominance in conflicting directions. We detect no phylogenetic signal nor an effect of parental genetic distance on dominance or mismatch. Using data from an experimental field planting of recombinant hybrid sunflowers--where there is among-individual variation in dominance and mismatch due to segregation of divergent alleles--we illustrate that pairwise parent-bias improves fitness while mismatch reduces fitness. Importantly, the effect of mismatch on fitness was stronger than that of pairwise parent-bias. In sum, our study has three major conclusions. First, hybrids between ecologically divergent natural populations are typically not phenotypically intermediate but rather exhibit substantial mismatch while also resembling one parent more than the other. Second, dominance and mismatch are likely determined by population-specific processes rather than general rules. Finally, selection against hybrids likely results from both selection against somewhat intermediate phenotypes and against mismatched trait combinations.

evolutionary biology↗

bioRxiv2019

Evidence that pleiotropic alleles underlie adaptive divergence between natural populations

The alleles used for adaptation can pleiotropically affect traits under stabilizing selection, and compensatory alleles can be favoured by selection to counteract such deleterious pleiotropy. Such compensatory alleles can segregate in interpopulation hybrids, causing segregation variance for traits where parents have the same phenotype. If adaptation typically involves pleiotropy and compensation, then the segregation variance for traits under stabilizing selection is expected to increase with the magnitude of adaptive phenotypic divergence between parents. This prediction has not been tested empirically, and I gathered data from experimental hybridization studies to evaluate it. I found that pairs of parents which are more phenotypically divergent beget hybrids with more segregation variance in traits where the parents do not differ. This result suggests that adaptive divergence between pairs of natural populations proceeds via pleiotropy and compensation, and that potentially deleterious transgressive segregation variance accumulates systematically as populations diverge.

evolutionary biology↗

bioRxiv2019

Multivariate phenotypic divergence along an urbanization gradient

A growing body of evidence suggests that natural populations can evolve to better tolerate the novel environmental conditions associated with urban areas. Invariably, studies of adaptive divergence in urban areas examine only one or a few traits at a time from populations residing only at the most extreme urban and nonurban habitats. Thus, whether urbanization is driving divergence in many traits simultaneously in a manner that varies with the degree of urbanization remains unclear. To address this gap, we generated seed families of white clover (Trifolium repens) collected from 27 populations along an urbanization gradient in Toronto, Canada, and grew them up to measure multiple phenotypic traits in a common garden. Overall, urban populations had later phenology and germination, larger flowers, thinner stolons, reduced cyanogenesis, greater biomass, and were more attractive to pollinators. Pollinator observations revealed near complete turnover between urban and nonurban sites, which may explain some of the observed divergence in floral traits and phenology. Our results suggest that adaptation to urban environments involves multiple organismal traits.

evolutionary biology↗

bioRxiv2019

Predicting the strength of urban-rural clines in a Mendelian polymorphism along a latitudinal gradient

Cities are emerging as models for addressing the fundamental question of whether populations evolve in parallel to similar environments. Here, we examine the environmental factors that drive parallel evolutionary urban-rural clines in a Mendelian trait -- the cyanogenic antiherbivore defense of white clover (Trifolium repens). We sampled over 700 urban and rural clover populations across 16 cities along a latitudinal transect in eastern North America. In each population, we quantified the frequency of genotypes that produce hydrogen cyanide (HCN), and in a subset of the cities we estimated the frequency of the alleles at the two genes (CYP79D15 and Li) that epistatically interact to produce HCN. We then tested the hypothesis that winter environmental conditions cause the evolution of clines in HCN by comparing the strength of clines among cities located along a gradient of winter temperatures and frost exposure. Overall, half of the cities exhibited urban-rural clines in the frequency of HCN, whereby urban populations evolved lower HCN frequencies. The weakest clines in HCN occurred in cities with the lowest temperatures but greatest snowfall, supporting the hypothesis that snow buffers plants against winter frost and constrains the formation of clines. By contrast, the strongest clines occurred in the warmest cities where snow and frost are rare, suggesting that alternative selective agents are maintaining clines in warmer cities. Additionally, some clines were driven by evolution at only CYP79D15, consistent with stronger and more consistent selection on this locus than on Li. Together, our results demonstrate that both the agents and targets of selection vary across cities and highlight urban environments as large-scale models for disentangling the causes of parallel evolution in nature.\n\nImpact SummaryUnderstanding whether independent populations evolve in the same way (i.e., in parallel) when subject to similar environments remains an important problem in evolutionary biology. Urban environments are a model for addressing the extent of parallel evolution in nature due to their convergent environments (e.g. heat islands, pollution, fragmentation), such that two distant cities are often more similar to one another than either is to nearby nonurban habitats. In this paper, we used white clover (Trifolium repens) as a model to study the drivers of parallel evolution in response to urbanization. We collected >11,000 plants from urban and rural habitats across 16 cities in eastern North America to examine how cities influence the evolution of a Mendelian polymorphism for an antiherbivore defense trait - hydrogen cyanide (HCN). This trait had previously been shown to exhibit adaptive evolution to winter temperature gradients at continental scales. Here we tested the hypothesis that winter environmental conditions cause changes in the frequency of HCN between urban and rural habitats. We found that half of all cities had lower frequency of HCN producing genotypes relative to rural habitats, demonstrating that cities drive parallel losses of HCN in eastern North America. We then used environmental data to understand why cities vary in the extent to which they drive reduction in HCN frequencies. The warmest cities showed the greatest reductions in HCN frequencies in urban habitats, while colder, snowier cities showed little change in HCN between urban and rural habitats. This suggests that snow weakens the strength of natural selection against HCN in cities. However, it additionally suggests alternative ecological or evolutionary mechanisms drive the strong differences in HCN between urban and rural habitats in the warmest cities. Overall, our work highlights urban environments as powerful, large-scale models for disentangling the causes of parallel and non-parallel evolution in nature.

evolutionary biology↗