The extent of introgression between incipient Clarkia species is determined by temporal environmental variation and mating system.

Introgression is pervasive across the tree of life but varies across taxa, geography, and genomic regions. However, the factors modulating this variation and how they may be affected by global change are not well understood. Here, we used 200 genomes and a 15-y site-specific environmental dataset to investigate the effects of environmental variation and mating system divergence on the magnitude of introgression between a recently diverged outcrosser-selfer pair of annual plants in the genus Clarkia. These sister taxa diverged very recently and subsequently came into secondary sympatry where they form replicated contact zones. Consistent with observations of other outcrosser-selfer pairs, we found that introgression was asymmetric between taxa, with substantially more introgression from the selfer to the outcrosser. This asymmetry was caused by a bias in the direction of initial F1 hybrid formation and subsequent backcrossing. We also found extensive variation in the outcrosser's admixture proportion among contact zones, which was predicted nearly entirely by interannual variance in spring precipitation. Greater fluctuations in spring precipitation resulted in higher admixture proportions, likely mediated by the effects of spring precipitation on the expression of traits that determine premating reproductive isolation. Climate-driven hybridization dynamics may be particularly affected by global change, potentially reshaping species boundaries and adaptation to novel environments.

Reproductive strategies and their consequences for divergence, gene flow, and genetic diversity in three taxa of Clarkia.

Differences in reproductive strategies can have important implications for macro- and micro-evolutionary processes. We used a comparative approach through a population genetics lens to evaluate how three distinct reproductive strategies shape patterns of divergence among as well as gene flow and genetic diversity within three closely related taxa in the genus Clarkia. One taxon is a predominantly autonomous self-fertilizer and the other two taxa are predominantly outcrossing but vary in the primary pollinator they attract. In genotyping populations using genotyping-by-sequencing and comparing loci shared across taxa, our results suggest that differences in reproductive strategies in part promote evolutionary divergence among these closely related taxa. Contrary to expectations, we found that the selfing taxon had the highest levels of heterozygosity but a low rate of polymorphism. The high levels of fixed heterozygosity for a subset of loci suggests this pattern is driven by the presence of structural rearrangements in chromosomes common in other Clarkia taxa. In evaluating patterns within taxa, we found a complex interplay between reproductive strategy and geographic distribution. Differences in the mobility of primary pollinators did not translate to a difference in rates of genetic diversity and gene flow within taxa - a pattern likely due to one taxon having a patchier distribution and a less temporally and spatially reliable pollinator. Taken together, this work advances our understanding of the factors that shape gene flow and the distribution of genetic diversity within and among closely related taxa.

Rapid evolution during climate change: demographic and genetic constraints on adaptation to severe drought.

Populations often vary in their evolutionary responses to a shared environmental perturbation. A key hurdle in building more predictive models of rapid evolution is understanding this variation-why do some populations and traits evolve while others do not? We combined long-term demographic and environmental data, estimates of quantitative genetic variance components, a resurrection experiment and individual-based evolutionary simulations to gain mechanistic insights into contrasting evolutionary responses to a severe multi-year drought. We examined five traits in two populations of a native California plant, Clarkia xantiana, at three time points over 7 years. Earlier flowering phenology evolved in only one of the two populations, though both populations experienced similar drought severity and demographic declines and were estimated to have considerable additive genetic variance for flowering phenology. Pairing demographic and experimental data with evolutionary simulations suggested that while seed banks in both populations probably constrained evolutionary responses, a stronger seed bank in the non-evolving population resulted in evolutionary stasis. Gene flow through time via germ banks may be an important, underappreciated control on rapid evolution in response to extreme environmental perturbations.

Genome-wide investigation of toll-like receptor genes (TLRs) in Procambarus clarkia and their expression pattern in response to black may disease.

As a crucial component of pattern-recognition receptors (PRRs) that recognizing pathogen-associated molecular patterns (PAMPs) and defending against invading pathogens, the Toll-like receptors (TLRs) have been paid extensive attention. While the identification and functional roles of TLRs in innate immunity have been reported in a plenty of organisms, the systematic knowledge of TLRs is still lacking in the red swamp crayfish (Procambarus clarkia). In current study, a total of 7 tlr genes were identified in P. clarkia based on the published transcriptome and genome data. The PcTLRs length varied from 939 to 1517aa and contain typical domains of TLR protein, including transmembrane region, varied LRR and TIR domains. 7 Pctlr genes were distributed in 5 chromosomes and 2 scaffolds. The expression pattern of different Pctlr genes in different tissues (hepatopancreas, gill and muscle) and in response to black may disease (BMD) showed significant difference. In addition, 5 proteins that might interact with PcTLR-2 were predicted, among them the expression pattern of dorsal and relish was consistent with Pctlr-2 in three tissues, while the other genes were not. The PcTLR-2-Dorsal/Relish pathway might play crucial roles in response to BMD infection. The results provided a theoretical foundation for further studies on the molecular mechanisms of TLRs in BMD infection in the red swamp crayfish and provided reference for the research of other crustacean species.

Effect of dietary replacement of fish meal by poultry by-product meal on the growth performance, immunity, and intestinal health of juvenile red swamp crayfish, procambarus clarkia.

The present study was conducted to investigate the dietary replacement of fish meal with poultry by-product meal (PBM) on the growth performance, immunity, antioxidant properties, and intestinal health of red swamp crayfish (Procambarus clarkia). A diet containing 20% fish meal (FM) and complex plant ingredients as the main protein resources was set as the FM group (crude protein 32%, crude lipid 6%). Four diets replacing 25%, 50%, 75%, and 100% fish meal of the FM diet with PBM were set as the PBM25, PBM50, PBM75, and PBM100 groups, respectively. Compared to the FM group, the PBM100 diet significantly decreased growth performance and feed utilization of crayfish, while markedly increasing the activity of serum aspartate aminotransferase. The immune response was depressed in crayfish fed the PBM100 diet as the activities of serum lysozyme and phenoloxidase, gene expression of anti-lipopolysaccharide factors (alf), cyclophilin A (cypa), crustin, and hemocyanin-1 (hep-1) in hepatopancreas were remarkably decreased. The activities of antioxidases and expression of antioxidant-relevant genes in the hepatopancreas were not influenced by PBM inclusion. Crayfish fed different diets exhibited no obvious symptoms of enteritis, but the PBM100 diet destructed intestinal morphology by significantly decreasing the average length of longitudinal ridges. The α-diversity and overall community structure were not significantly influenced but variations were found in the relative abundance of some genera by PBM inclusion. In summary, CAP could successfully replace 75% dietary FM in a basal diet containing 20% fish meal, while higher CAP level compromised growth performance, immunity, and intestinal histology of crayfish.

Context-dependent concordance between physiological divergence and phenotypic selection in sister taxa with contrasting phenology and mating systems.

PREMISE: The study of phenotypic divergence of, and selection on, functional traits in closely related taxa provides the opportunity to detect the role of natural selection in driving diversification. If the strength or direction of selection in field populations differs between taxa in a pattern that is consistent with the phenotypic difference between them, then natural selection reinforces the divergence. Few studies have sought evidence for such concordance for physiological traits. METHODS: Herbarium specimen records were used to detect phenological differences between sister taxa independent of the effects on flowering time of long-term variation in the climate across collection sites. In the field, physiological divergence in photosynthetic rate, transpiration rate, and instantaneous water-use efficiency were recorded during vegetative growth and flowering in 13 field populations of two taxon pairs of Clarkia, each comprising a self-pollinating and a outcrossing taxon. RESULTS: Historically, each selfing taxon flowered earlier than its outcrossing sister taxon, independent of the effects of local long-term climatic conditions. Sister taxa differed in all focal traits, but the degree and (in one case) the direction of divergence depended on life stage. In general, self-pollinating taxa had higher gas exchange rates, consistent with their earlier maturation. In 6 of 18 comparisons, patterns of selection were concordant with the phenotypic divergence (or lack thereof) between sister taxa. CONCLUSIONS: Patterns of selection on physiological traits measured in heterogeneous conditions do not reliably reflect divergence between sister taxa, underscoring the need for replicated studies of the direction of selection within and among taxa.

Emission Rates of Species-Specific Volatiles Vary across Communities of Clarkia Species: Evidence for Multimodal Character Displacement.

AbstractA current frontier of character displacement research is to determine whether displacement occurs via multiple phenotypic pathways and varies across communities with different species compositions. Here, we conducted the first test for context-dependent character displacement in multimodal floral signals by analyzing variation in floral scent in a system that exhibits character displacement in flower size and that has multiple types of sympatric communities. In a greenhouse common garden experiment, we measured quantitative variation in volatile emission rates of the progeny of two species of Clarkia from replicated parental communities that contain one, two, or four Clarkia species. The first two axes of a constrained correspondence analysis, which explained 24% of the total variation in floral scent, separated the species and community types. Of the 23 compounds that were significantly correlated with these axes, nine showed patterns consistent with character displacement. Two compounds produced primarily by C. unguiculata and two compounds produced primarily by C. cylindrica were emitted in higher amounts in sympatry. Character displacement in some volatiles varied across sympatric parental communities and occurred in parallel with displacement in flower size, demonstrating that this evolutionary process can be context dependent and may occur through multiple pathways.

Phylogenomic analysis does not support a classic but controversial hypothesis of progenitor-derivative origins for the serpentine endemic Clarkia franciscana.

Budding speciation involves isolation of marginal populations at the periphery of a species range and is thought to be a prominent mode of speciation in organisms with low dispersal and/or strong local adaptation among populations. Budding speciation is typically evidenced by abutting, asymmetric ranges of ecologically divergent sister species and low genetic diversity in putative budded species. Yet these indirect patterns may be unreliable, instead caused by postspeciation processes such as range or demographic shifts. Nested phylogenetic relationships provide the most conclusive evidence of budding speciation. A putative case of budding speciation in the serpentine endemic Clarkia franciscana and two closely related widespread congeners was studied by Harlan Lewis, Peter Raven, Leslie Gottlieb, and others over a 20-year period, yet the origin of C. franciscana remains controversial. Here, we reinvestigate this system with phylogenomic analyses to determine whether C. franciscana is a recently derived budded species, phylogenetically nested within one of the other two putative progenitor species. In contrast to the hypothesized pattern of relatedness among the three Clarkia species, we find no evidence for recent budding speciation. Instead, the data suggest the three species diverged simultaneously. We urge caution in using contemporary range patterns to infer geographic modes of speciation.

A greenhouse experiment partially supports inferences of ecogeographic isolation from niche models of Clarkia sister species.

PREMISE: Ecogeographic isolation, or geographic isolation caused by ecological divergence, is thought to be of primary importance in speciation, yet is difficult to demonstrate and quantify. To determine whether distributions are limited by divergent adaptation or historical contingency, the gold standard is to reciprocally transplant species between their geographic ranges. Alternatively, ecogeographic isolation is inferred from species distribution models and niche divergence tests using widely available environmental and occurrence data. METHODS: We tested for ecogeographic isolation between two sister species of California annual wildflowers, Clarkia concinna and C. breweri, with a hybrid approach. We used niche models to predict water availability as the major axis of ecological divergence and then tested that with a greenhouse experiment. Specifically, we manipulated water availability in field soils for two populations of each species and predicted higher fitness in conditions representing home habitats to those representing the environment of each's sister species. RESULTS: Water availability and soil representing C. concinna generally increased both species' fitness. Thus, water and soil may indeed limit C. concinna from colonizing the range of C. breweri, but not vice versa. We suggest that the competitive environment and pollinator availability, which are not directly captured with either approach, may be key biotic factors correlated with climate that contribute to unexplained ecogeographic isolation for C. breweri. CONCLUSIONS: Ours is a valuable approach to assessing ecogeographic isolation, in that it balances feasibility with model validation, and our results have implications for species distribution modeling efforts geared toward predicting climate change responses.

Transcriptome analysis reveals antioxidant defense mechanisms in the red swamp crayfish Procambarus clarkia after exposure to chromium.

Chromium (Cr) as a chromate anion has a strong redox capacity that seriously threatens the ecological environment and human health. Cr can contaminate water and impart toxicity to aquatic species. Procambarus clarkii is an important food source that once represented a large proportion of the aquaculture industry due to its rapid reproduction and high economic value. However, there have been reports on the death of P. clarkii due to heavy metal pollution. The underlying mechanism regarding heavy metal toxicity was studied in this paper. The transcriptome data of hemocytes extracted from P. clarkii injected with Cr were analyzed by high-throughput sequencing and compared to the control group. In total, 48,128,748 clean reads were obtained in the treatment group and 56,480,556 clean reads were obtained in the control group. The reads were assembled using Trinity and the identified unigenes were then annotated. Then, 421 differentially-expressed genes (DEGs) were found, 170 of which were upregulated and 251 downregulated. Many of these genes were found to be related to glutathione metabolism and transportation. The glutathione metabolic pathway of P. clarkii was thus activated by Cr exposure to detoxify and maintain body function. Validation of DEGs with quantitative real-time PCR confirms the changes in gene expression. Thus, this study provides data supporting a glutathione-focused response of P. clarkii to exposure to heavy metals.

Ancient Gene Duplications, Rather Than Polyploidization, Facilitate Diversification of Petal Pigmentation Patterns in Clarkia gracilis (Onagraceae).

It has been suggested that gene duplication and polyploidization create opportunities for the evolution of novel characters. However, the connections between the effects of polyploidization and morphological novelties have rarely been examined. In this study, we investigated whether petal pigmentation patterning in an allotetraploid Clarkia gracilis has evolved as a result of polyploidization. Clarkia gracilis is thought to be derived through a recent polyploidization event with two diploid species, C. amoena huntiana and an extinct species that is closely related to C. lassenensis. We reconstructed phylogenetic relationships of the R2R3-MYBs (the regulators of petal pigmentation) from two subspecies of C. gracilis and the two purported progenitors, C. a. huntiana and C. lassenensis. The gene tree reveals that these R2R3-MYB genes have arisen through duplications that occurred before the divergence of the two progenitor species, that is, before polyploidization. After polyploidization and subsequent gene loss, only one of the two orthologous copies inherited from the progenitors was retained in the polyploid, turning it to diploid inheritance. We examined evolutionary changes in these R2R3-MYBs and in their expression, which reveals that the changes affecting patterning (including expression domain contraction, loss-of-function mutation, cis-regulatory mutation) occurred after polyploidization within the C. gracilis lineages. Our results thus suggest that polyploidization itself is not necessary in producing novel petal color patterns. By contrast, duplications of R2R3-MYB genes in the common ancestor of the two progenitors have apparently facilitated diversification of petal pigmentation patterns.

Evolutionary divergence of potential drought adaptations between two subspecies of an annual plant: Are trait combinations facilitated, independent, or constrained?

PREMISE: Whether drought-adaptation mechanisms tend to evolve together, evolve independently, or evolve constrained by genetic architecture is incompletely resolved, particularly for water-relations traits besides gas exchange. We addressed this issue in two subspecies of Clarkia xantiana (Onagraceae), California winter annuals that separated approximately 65,000 years ago and are adapted, partly by differences in flowering time, to native ranges differing in precipitation. METHODS: In these subspecies and in recombinant inbred lines (RILs) from a cross between them, we scored traits related to drought adaptation (timing of seed germination and of flowering, succulence, pressure-volume curve variables) in common environments. RESULTS: The subspecies native to more arid environments (parviflora) exhibited slower seed germination in saturated conditions, earlier flowering, and greater succulence, likely indicating superior drought avoidance, drought escape, and dehydration resistance via water storage. The other subspecies (xantiana) had lower osmotic potential at full turgor and lower water potential at turgor loss, implying superior dehydration tolerance. Genetic correlations among RILs suggest facilitated evolution of some trait combinations and independence of others. Where genetic correlations exist, subspecies differences fell along them, with the exception of differences in succulence and turgor loss point. In that case, subspecies difference overcame genetic correlations, possibly reflecting strong selection and/or antagonistic genetic correlations with other traits. CONCLUSIONS: Clarkia xantiana subspecies' differ in multiple mechanisms of drought adaptation. Genetic architecture generally does not seem to have constrained the evolution of these mechanisms, and it may have facilitated the evolution of some of trait combinations.

R2R3-MYB genes control petal pigmentation patterning in Clarkia gracilis ssp. sonomensis (Onagraceae).

Petal pigmentation patterning is widespread in flowering plants. The genetics of these pattern elements has been of great interest for understanding the evolution of phenotypic diversification. Here, we investigate the genetic changes responsible for the evolution of an unpigmented petal element on a colored background. We used transcriptome analysis, gene expression assays, cosegregation in F2 plants and functional tests to identify the gene(s) involved in petal coloration in Clarkia gracilis ssp. sonomensis. We identified an R2R3-MYB transcription factor (CgsMYB12) responsible for anthocyanin pigmentation of the basal region ('cup') in the petal of C. gracilis ssp. sonomensis. A functional mutation in CgsMYB12 creates a white cup on a pink petal background. Additionally, we found that two R2R3-MYB genes (CgsMYB6 and CgsMYB11) are also involved in petal background pigmentation. Each of these three R2R3-MYB genes exhibits a different spatiotemporal expression pattern. The functionality of these R2R3-MYB genes was confirmed through stable transformation of Arabidopsis. Distinct spatial patterns of R2R3-MYB expression have created the possibility that pigmentation in different sections of the petal can evolve independently. This finding suggests that recent gene duplication has been central to the evolution of petal pigmentation patterning in C. gracilis ssp. sonomensis.

Plant-soil interactions limit lifetime fitness outside a native plant's geographic range margin.

Plant species' distributions are often thought to overwhelmingly reflect their climatic niches. However, climate represents only a fraction of the n-dimensional environment to which plant populations adapt, and studies are increasingly uncovering strong effects of nonclimatic factors on species' distributions. We used a manipulative, factorial field experiment to quantify the effects of soil environment and precipitation (the putatively overriding climatic factor) on plant lifetime fitness outside the geographic range boundary of a native California annual plant. We grew plants outside the range edge in large mesocosms filled with soil from either within or outside the range, and plants were subjected to either a low (ambient) or high (supplemental) spring precipitation treatment. Soil environment had large effects on plant lifetime fitness that were similar in magnitude to the effects of precipitation. Moreover, mean fitness of plants grown with within-range soil in the low precipitation treatment approximated that of plants grown with beyond-range soil in the high precipitation treatment. The positive effects of within-range soil persisted in the second, wetter year of the experiment, though the magnitude of the soil effect was smaller than in the first, drier year. These results are the first we know of to quantify the effects of edaphic variation on plant lifetime fitness outside a geographic range limit and highlight the need to include factors other than climate in models of species' distributions.

Microbes, mutualism, and range margins: testing the fitness consequences of soil microbial communities across and beyond a native plant's range.

Interactions between plants and soil fungi and bacteria are ubiquitous and have large effects on individual plant fitness. However, the degree to which spatial variation in soil microbial communities modulates plant species' distributions remains largely untested. Using the California native plant Clarkia xantiana ssp. xantiana we paired glasshouse and field reciprocal transplants of plant populations and soils to test whether plant-microbe interactions affect the plant's geographic range limit and whether there is local adaptation between plants and soil microbe communities. In the field and glasshouse, one of the two range interior inocula had a positive effect on plant fitness. In the field, this benefit was especially pronounced at the range edge and beyond, suggesting possible mutualist limitation. In the glasshouse, soil inocula from beyond-range tended to increase plant growth, suggesting microbial enemy release beyond the range margin. Amplicon sequencing revealed stark variation in microbial communities across the range boundary. Plants dispersing beyond their range limit are likely to encounter novel microbial communities. In C. x. xantiana, our results suggest that range expansion may be facilitated by fewer pathogens, but could also be hindered by a lack of mutualists. Both negative and positive plant-microbe interactions will likely affect contemporary range shifts.

Digest: Floral evolution through the lens of the community context.

How do emergent properties of natural plant communities affect floral evolution? In this issue, Eisen et al. explored this question by studying selection on floral traits in natural communities of Clarkia species. They found that two community properties, namely congeneric species richness and floral density (of conspecifics and heterospecifics), influenced the patterns of selection, although not through the expected means of pollinator-mediated selection. Instead, the authors suggest that additional factors like competition and facilitation between plants are responsible. The results support the hypothesis that, beside pollinators, other factors of the community context can also determine floral evolution.

The opportunity for outcrossing varies across the geographic range of the primarily selfing Clarkia xantiana ssp. parviflora.

PREMISE: The timing of self-fertilization has potentially important consequences for the trajectory of mating system evolution, the opportunity for outcrossing, and the maintenance of genetic variation in populations. For primarily selfing taxa, it remains poorly understood as to how floral variation influences the opportunity for outcrossing and whether those attributes vary among populations across geographic ranges. METHODS: We examined variation in floral traits (herkogamy, protandry, flower size, stigma stage at anthesis, timing of stigma receptivity) in seven populations of Clarkia xantiana ssp. parviflora, a primarily selfing taxon, spanning from the western to eastern margins of its distribution. We also performed experimental emasculations and pollinations (followed by stigma severing) to quantify the extent of opportunities for outcrossing across flower development. RESULTS: There was marked among-population variation in all floral traits, particularly between far eastern and western populations. Emasculation experiments showed that the eastern populations had minimal autonomous selfing, but western populations had high rates of selfing within 24 h after anthesis. Population variation in autofertility was significantly predicted by floral trait variation, especially protandry and petal size. CONCLUSIONS: Greater protandry both extended the time over which outcrossing could potentially occur and reduced the probability of autonomous selfing, suggesting that there may be a tradeoff that results in fitness loss when pollinator visits are not common. The east-west pattern of differentiation in some floral traits parallels that of postglacial range expansion, suggesting that selection on the mating system may have been strong in the process of range expansion.

Pathogenicity of Aeromonas hydrophila causing mass mortalities of Procambarus clarkia and its induced host immune response.

Outbreaks of mass mortalities among cultured Procambarus clarkia occurred in a commercial hatchery during the spring of 2019 in Jiangsu province of China. Here, we exploit the pathogenicity and immune response of Aeromonas hydrophila (GPC1-2), which was isolated from diseased P. clarkia. Crayfish challenged showed similar pathological signs to the naturally diseased P. clarkia, lethal dose 50% (LD50) of the strain GPC1-2 to P. clarkia was 3.8 × 106 CFU/mL. Detection of virulence-associated genes by PCR indicated that the strain GPC1-2 carried hlyA, aerA, alt, ast, act, aha, ahp, ahpA, and ahpB. Histopathological analysis of hepatopancreas revealed that the hepatic tubule lumen and the gap between the hepatic tubules became larger, and the brush border disappeared in the P. clarkia infected by GPC1-2. Quantitive real-time PCR (qRT-PCR) was undertaken to measure mRNA expression levels for six immune-related genes in P. clarkia after A. hydrophila infection. The expression level of proPO, NOS, ALF1, TLR2, PX, and AST were detected in hemolymph, hepatopancreas, gill and intestine tissues, and clear transcriptional activation of these genes were observed in the infected individuals. These results revealed pathogenicity of A. hydrophila and its activation of host immune response, which will provide a scientific reference for the breeding and disease prevention in P. clarkia culture.

Floral density and co-occurring congeners alter patterns of selection in annual plant communities.

Although the evolution and diversification of flowers is often attributed to pollinator-mediated selection, interactions between co-occurring plant species can alter patterns of selection mediated by pollinators and other agents. The extent to which both floral density and congeneric species richness affect patterns of net and pollinator-mediated selection on multiple co-occurring species in a community is unknown and is likely to depend on whether co-occurring plants experience competition or facilitation for reproduction. We conducted an observational study of selection on four species of Clarkia (Onagraceae) and tested for pollinator-mediated selection on two Clarkia species in communities differing in congeneric species richness and local floral density. When selection varied with community context, selection was generally stronger in communities with fewer species, where local conspecific floral density was higher, and where local heterospecific floral density was lower. These patterns suggest that intraspecific competition at high densities and interspecific competition at low densities may affect the evolution of floral traits. However, selection on floral traits was not pollinator mediated in Clarkia cylindrica or Clarkia xantiana, despite variation in pollinator visitation and the extent of pollen limitation across communities for C. cylindrica. As such, interactions between co-occurring species may alter patterns of selection mediated by abiotic agents of selection.

Maladaptation beyond a geographic range limit driven by antagonistic and mutualistic biotic interactions across an abiotic gradient.

Species' geographic range limits often result from maladaptation to the novel environments beyond the range margin. However, we rarely know which aspects of the n-dimensional environment are driving this maladaptation. Especially of interest is the influence of abiotic versus biotic factors in delimiting species' distributions. We conducted a 2-year reciprocal transplant experiment involving manipulations of the biotic environment to explore how spatiotemporal gradients in precipitation, fatal mammalian herbivory, and pollination affected lifetime fitness within and beyond the range of the California annual plant, Clarkia xantiana ssp. xantiana. In the first, drier year of the experiment, fitness outside the range edge was limited mainly by low precipitation, and there was some evidence for local adaptation within the range. In the second, wetter year, we did not observe abiotic limitations to plant fitness outside the range; instead biotic interactions, especially herbivory, limited fitness outside the range. Together, protection from herbivory and supplementation of pollen resulted in three- to sevenfold increases in lifetime fitness outside the range margin in the abiotically benign year. Overall, our work demonstrates the importance of biotic interactions, particularly as they interact with the abiotic environment, in determining fitness beyond geographic range boundaries.