Urban ecosystems are an increasingly dominant feature of terrestrial landscapes. While evidence that species can adapt to urban environments is accumulating, the mechanisms through which urbanization imposes natural selection on populations are poorly understood. The identification of adaptive phenotypic changes (i.e. clines) along urbanization gradients would facilitate our understanding of the selective factors driving adaptation in cities. Here, we test for phenotypic clines in urban ecosystems by sampling the frequency of a Mendelian-inherited trait-cyanogenesis-in white clover (Trifolium repens L.) populations along urbanization gradients in four cities. Cyanogenesis protects plants from herbivores, but reduces tolerance to freezing temperatures. We found that the frequency of cyanogenic plants within populations decreased towards the urban centre in three of four cities. A field experiment indicated that spatial variation in herbivory is unlikely to explain these clines. Rather, colder minimum winter ground temperatures in urban areas compared with non-urban areas, caused by reduced snow cover in cities, may select against cyanogenesis. In the city with no cline, high snow cover might protect plants from freezing damage in the city centre. Our study suggests that populations are adapting to urbanization gradients, but regional climatic patterns may ultimately determine whether adaptation occurs.

Biological nitrogen fixation (BNF) by microorganisms associated with cryptogamic covers, such as cyanolichens and bryophytes, is a primary source of fixed nitrogen in pristine, high-latitude ecosystems. On land, low molybdenum (Mo) availability has been shown to limit BNF by the most common form of nitrogenase (Nase), which requires Mo in its active site. Vanadium (V) and iron-only Nases have been suggested as viable alternatives to countering Mo limitation of BNF; however, field data supporting this long-standing hypothesis have been lacking. Here, we elucidate the contribution of vanadium nitrogenase (V-Nase) to BNF by cyanolichens across a 600-km latitudinal transect in eastern boreal forests of North America. Widespread V-Nase activity was detected (∼15–50% of total BNF rates), with most of the activity found in the northern part of the transect. We observed a 3-fold increase of V-Nase contribution during the 20-wk growing season. By including the contribution of V-Nase to BNF, estimates of new N input by cyanolichens increase by up to 30%. We find that variability in V-based BNF is strongly related to Mo availability, and we identify a Mo threshold of ∼250 ng·glichen−1 for the onset of V-based BNF. Our results provide compelling ecosystem-scale evidence for the use of the V-Nase as a surrogate enzyme that contributes to BNF when Mo is limiting. Given widespread findings of terrestrial Mo limitation, including the carbon-rich circumboreal belt where global change is most rapid, additional consideration of V-based BNF is required in experimental and modeling studies of terrestrial biogeochemistry.

Moss‐associated cyanobacteria nitrogen (N2‐) fixation can contribute to support moss growth and constitutes a major source of new N in boreal forest ecosystems. The biomass of moss‐colonizing cyanobacteria and their N2‐fixation are usually considered linearly correlated. Yet, recent evidence has shown that cyanobacterial biomass and N2‐fixation can be decoupled, suggesting that they are not necessary affected by the same environmental and ecological drivers. Climate and nutrients were reported as affecting moss‐associated N2‐fixation, with equivocal results, whereas drivers of moss cyanobacterial biomass remain unclear. In addition, these drivers are often determined through manipulative experiments (e.g. fertilization and incubation) and remain to be validated with complementary observational studies to help us better understand future impacts of global change on the moss–cyanobacteria symbiosis. We hypothesized that moss‐associated cyanobacterial biomass is controlled in situ by factors affecting bacterial growth, whereas N2‐fixation is controlled by factors affecting enzymatic reactions. Using random forests, Spearman correlations and linear mixed‐effects models, we determined the main drivers of cyanobacterial biomass and N2‐fixation of two feather moss species, which were collected over 3 years along a 1000‐km latitudinal transect in the eastern Canadian boreal forest. We found that temperature, precipitation and phosphorus were the main positive drivers of moss cyanobacterial biomass and that temperature and molybdenum were the main positive drivers of N2‐fixation. Vanadium was a negative driver of N2‐fixation, suggesting the use of alternative nitrogenases by cyanobacteria. Both cyanobacterial biomass and N2‐fixation were strongly influenced by the moss species and were negatively correlated with moss C:N stoichiometry, highlighting the role of N2‐fixation in moss N enrichment. Synthesis. We identified for the first time some environmental drivers of moss‐associated cyanobacterial biomass and showed that they contrast with the drivers of N2‐fixation, which should be considered in further research and confirmed in other experimental settings. This is an important advance in our knowledge of moss–cyanobacteria associations, which would greatly help in better predicting the impacts of global change on this symbiosis and on nitrogen inputs in boreal forest ecosystems.

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In the boreal forest, cyanobacteria can establish associations with feather moss and realize the biological nitrogen fixation (BNF) reaction, consisting in the reduction of atmospheric dinitrogen into bioavailable ammonium. In this ecosystem, moss-associated cyanobacteria are the main contributors to BNF by contributing up to 50% of new N input. Current environmental changes driven by anthropogenic activities will likely affect cyanobacteria activity (i.e., BNF) and populations inhabiting mosses, leading to potential important consequences for the boreal forest. Several methods are available to efficiently measure BNF activity, but quantifying cyanobacteria biomass associated with moss is challenging because of the difficulty to separate bacteria colonies from the host plant. Attempts to separate cyanobacteria by shaking or sonicating in water were shown to be poorly efficient and repeatable. The techniques commonly used, microscopic counting and quantitative PCR (qPCR) are laborious and time-consuming. In aquatic and marine ecosystems, phycocyanin (PC), a photosynthesis pigment produced by cyanobacteria, is commonly used to monitor cyanobacteria biomass. In this study, we tested if PC extraction and quantification can be used to estimate cyanobacteria quantity inhabiting moss. We report that phycocyanin can be easily extracted from moss by freeze/thaw disturbance of cyanobacteria cells and can be quickly and efficiently measured by spectrofluorometry. We also report that phycocyanin extraction is efficient (high recovery), repeatable (relative SD < 13%) and that no significant matrix effects were observed. As for aquatic systems, the main limitation of cyanobacteria quantification using phycocyanin is the difference of cellular phycocyanin content between cyanobacteria strains, suggesting that quantification can be impacted by cyanobacteria community composition. Nonetheless, we conclude that phycocyanin extraction and quantification is an easy, rapid, and efficient tool to estimate moss-associated cyanobacteria number.

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13Urban areas are a new and increasingly dominant feature of terrestrial landscapes that 14 dramatically alter environments. It is unclear whether wild populations can adapt to the 15 unique challenges presented by urbanization. To address this problem, we sampled the 16 frequency of a Mendelian-inherited trait-cyanogenesis-in white clover (Trifolium 17 repens L.) plants along urbanization gradients in four large cities. Cyanogenesis protects 18 plants from herbivores, but also reduces freezing tolerance. Plants evolved reduced 19 cyanogenesis with increasing proximity to the urban center in three of the four cities. In 20 an experiment, we demonstrate that gradients in herbivore pressure do not cause these 21 clines. Instead, urban areas experience relatively cold minimum winter ground 22 temperatures because of reduced snow cover within cities, which selects against 23 2 cyanogenesis. Together, our study demonstrates that wild populations exhibit parallel 24 adaptive evolution in response to urbanization, which likely facilitates the persistence of 25 these plants and promotes pollinator abundance and diversity. 26 27 Main Text 28 (hereafter Ac) and Li; Plants must have dominant alleles at both loci to produce HCN 47 . The two components are stored separately in cells to avoid self-48 toxicity, and form HCN when they are brought together following tissue damage. 49Previous research found that cyanogenesis is most frequent in populations at low latitudes 50 and elevations, where herbivory is often highest and temperatures are warmer (Daday 51 1954a). It has been hypothesized that herbivores select for cyanogenesis in these warm 52 environments (Hughes 1991; Kooyers and Olsen 2012). In cold climates, cyanogenesis is 53 selected against because freezing temperatures lyse cells and trigger HCN release, 54 causing self-toxicity in cyanogenic plants (Daday 1958;Hughes 1991). 55 Environmental change associated with urbanization may alter natural selection on 56 cyanogenesis. Whereas herbivores are not consistently affected by urbanization (Raupp et 57 al. 2010), air temperatures are generally warmer in urban areas relative to non-urban 58 areas (Landsberg 1981). This may cause urban populations to experience fewer freezing 59 events than non-urban populations during winter (Parris and Hazell 2005), and 60consequently weaken selection against cyanogenesis in cities. Thus, we predicted that 61 cyanogenesis would increase in frequency with increasing proximity to urban centers. We 62 surveyed 2379 plants from 121 populations of white clover along three 50-km transects 63 radiating outward from downtown Toronto, Canada (Fig. S1), and screened them for their 64 ability to produce HCN. To confirm that selection is acting on HCN, and not on Ac or Li 65 individually for unrelated functions, we also quantified the frequencies of the Ac and Li 66 alleles. 67 68 69 4 Results and Discussion 70We found consistent changes in the frequency of cyanogenesis along each transect, but 71 clines were in the opposite direction to our predi...