Support for Baker's law: Facultative self-fertilization ability decreases pollen limitation in experimental colonization.

PREMISE: The ability to self-fertilize is predicted to provide an advantage in colonization because a single individual can reproduce and establish a next generation in a new location regardless of the density of mates. While there is theoretical and correlative support for this idea, the strength of mate limitation as a selective agent has not yet been delineated from other factors that can also select for self-fertilization in colonization of new habitats. We used known mating-system variation in the American bellflower (Campanula americana) to explore how plants' ability to self-fertilize can mitigate density-dependent reproduction and impact colonization success. METHODS: We created experimental populations of single individuals or a small number of plants to emulate isolated colonization events. These populations were composed of plants that differed in their ability to self-fertilize. We compared pollen limitation of the single individuals to that of small populations. RESULTS: Experimental populations of plants that readily self-fertilize produced consistent seed numbers regardless of population size, whereas plants with lower ability to self-fertilize had density-dependent reproduction with greater seed production in small populations than in populations composed of a single individual. CONCLUSIONS: We experimentally isolated the effect of mate limitation in colonization and found that it can select for increased self-fertilization. We show the benefit of self-fertilization in colonization, which helps to explain geographic patterns of self-fertilization and shows support for Baker's law, a long-held hypothesis in the field of mating-system evolution.

Hybrid breakdown is elevated near the historical cores of a species' range.

New species form when they become reproductively isolated. A classic model of speciation posits that derived mutations appear in isolated populations and reduce fitness when combined in hybrids. While these Bateson-Dobzhansky-Muller incompatibilities are known to accumulate as populations diverge over time, they may also reflect the amount of standing genetic variation within populations. We analysed the fitness of F2 hybrids in crosses between 24 populations of a plant species (Campanula americana) with broad variation in standing genetic variation and genetic differentiation driven by post-glacial range expansions. Hybrid breakdown varied substantially and was strongest between populations near the historical cores of the species range where within-population genetic diversity was high. Nearly half of the variation in hybrid breakdown was predicted by the combined effects of standing genetic variation within populations, their pairwise genetic differentiation and differences in the climates they inhabit. Hybrid breakdown was enhanced between populations inhabiting distinct climates, likely reflecting local adaptation. Results support that the mutations causing hybrid breakdown, the raw material for speciation, are more common in long-inhabited areas of the species range. Genetic diversity harboured in refugial areas is thus an important source of incompatibilities critical to the speciation process.

Westward range expansion from middle latitudes explains the Mississippi River discontinuity in a forest herb of eastern North America.

It is often expected that temperate plants have expanded their geographical ranges northward from primarily southern refugia. Evidence for this hypothesis is mixed in eastern North American species, and there is increasing support for colonization from middle latitudes. We studied genome-wide patterns of variation in RADseq loci to test hypotheses concerning range expansion in a North American forest herb (Campanula americana). First, spatial patterns of genetic differentiation were determined. Then phylogenetic relationships and divergence times were estimated. Spatial signatures of genetic drift were also studied to identify the directionality of recent range expansion and its geographical origins. Finally, spatially explicit scenarios for the spread of plants across the landscape were compared, using variation in the population mutation parameter and Tajima's D. We found strong longitudinal subdivision, with populations clustering into groups west and east of the Mississippi River. While the southeastern region was probably part of a diverse Pleistocene refugium, there is little evidence that range expansion involved founders from these southern locales. Instead, declines in genetic diversity and the loss of rare alleles support a westward colonization wave from a middle latitude refugium near the southern Appalachian Mountains, with subsequent expansion from a Pleistocene staging ground in the Mississippi River Valley (0.51-1.27 million years ago). These analyses implicate stepping stone colonization from middle latitudes as an important mechanism of species range expansion in eastern North America. This study further demonstrates the utility of population genetics as a tool to infer the routes travelled by organisms during geographical range expansion.

Pollen colour morphs take different paths to fitness.

Colour phenotypes are often involved in communication and are thus under selection by species interactions. However, selection may also act on colour through correlated traits or alternative functions of biochemical pigments. Such forms of selection are instrumental in maintaining petal colour diversity in plants. Pollen colour also varies markedly, but the maintenance of this variation is little understood. In Campanula americana, pollen ranges from white to dark purple, with darker morphs garnering more pollinator visits and exhibiting elevated pollen performance under heat stress. Here, we generate an F2 population segregating for pollen colour and measure correlations with floral traits, pollen attributes and plant-level traits related to fitness. We determine the pigment biochemistry of colour variants and evaluate maternal and paternal fitness of light and dark morphs by crossing within and between morphs. Pollen colour was largely uncorrelated with floral traits (petal colour, size, nectar traits) suggesting it can evolve independently. Darker pollen grains were larger and had higher anthocyanin content (cyanidin and peonidin) which may explain why they outperform light pollen under heat stress. Overall, pollen-related fitness metrics were greater for dark pollen, and dark pollen sires generated seeds with higher germination potential. Conversely, light pollen plants produce 61% more flowers than dark, and 18% more seeds per fruit, suggesting a seed production advantage. Results indicate that light and dark morphs may achieve fitness through different means-dark morphs appear to have a pollen advantage whereas light morphs have an ovule advantage-helping to explain the maintenance of pollen colour variation.

The role of pollinator preference in the maintenance of pollen colour variation.

BACKGROUND AND AIMS: Pollinators often drive the evolution of floral traits, but their capacity to influence the evolution of pollen colour remains unclear. Pollen colour in Campanula americana is variable and displays a longitudinal cline from prevalence of deep purple in western populations to white and light-purple pollen in eastern populations. While selection for thermal tolerance probably underlies darker pollen in the west, factors contributing to the predominance of light pollen in eastern populations and the maintenance of colour variation within populations throughout the range are unknown. Here we examine whether pollinators contribute to the maintenance of pollen colour variation in C. americana. METHODS: In a flight cage experiment, we assessed whether Bombus impatiens foragers can use pollen colour as a reward cue. We then established floral arrays that varied in the frequency of white- and purple-pollen plants in two naturally occurring eastern populations. We observed foraging patterns of wild bees, totalling >1100 individual visits. KEY RESULTS: We successfully trained B. impatiens to prefer one pollen colour morph. In natural populations, the specialist pollinator, Megachile campanulae, displayed a strong and consistent preference for purple-pollen plants regardless of morph frequency. Megachile also exhibited a bias toward pollen-bearing male-phase flowers, and this bias was more pronounced for purple pollen. The other main pollinators, Bombus spp. and small bees, did not display pollen colour preference. CONCLUSIONS: Previous research found that Megachile removes twice as much pollen per visit as other bees and can deplete pollen from natural populations. Taken together, these results suggest that Megachile could reduce the reproductive success of plants with purple pollen, resulting in the prevalence of light-coloured pollen in eastern populations of C. americana. Our research demonstrates that pollinator preferences may play a role in the maintenance of pollen colour variation in natural populations.

Pollen limitation and autonomous selfing ability interact to shape variation in outcrossing rate across a species range.

PREMISE: Hermaphroditic plants commonly reproduce through a mixture of selfing and outcrossing. The degree to which outcrossing rates reflect the availability of outcross pollen, genetic differentiation in the ability to autonomously self-fertilize, or both is often unclear. Despite the potential for autonomy and the pollination environment to jointly influence outcrossing, this interaction is rarely studied. METHODS: We reviewed studies from the literature that tested whether the pollination environment or floral traits that cause autonomous selfing predict variation in outcrossing rate among populations. We also measured outcrossing rates in 23 populations of Campanula americana and examined associations with the pollination environment, autonomy, and their interaction. RESULTS: Our review revealed that traits that facilitate selfing were often negatively associated with outcrossing rates whereas most aspects of the pollination environment poorly predicted outcrossing. Populations of C. americana varied from mixed mating to highly outcrossing, but variation was unrelated to population size, density, pollen limitation, or autonomous selfing ability. Outcrossing rate was significantly influenced by an interaction between autonomous selfing ability and pollen limitation. Across highly autonomous populations, elevated pollen limitation was associated with reduced outcrossing, while there was no relationship for less autonomous populations. CONCLUSIONS: Both the ability to self autonomously and pollen limitation interact to shape outcrossing rates in C. americana. This work suggests that autonomy affords mating-system flexibility, though it is not ubiquitous in all populations across the species range. Interactions between traits influencing autonomy and pollen limitation are likely to explain variation in outcrossing rates among populations of flowering plants.

Linking pollinator efficiency to patterns of pollen limitation: small bees exploit the plant-pollinator mutualism.

Seemingly mutualistic relationships can be exploited, in some cases reducing fitness of the exploited species. In plants, the insufficient receipt of pollen limits reproduction. While infrequent pollination commonly underlies pollen limitation (PL), frequent interactions with low-efficiency, exploitative pollinators may also cause PL. In the widespread protandrous herb Campanula americana, visitation by three pollinators explained 63% of the variation in PL among populations spanning the range. Bumblebees and the medium-sized Megachile campanulae enhanced reproductive success, but small solitary bees exacerbated PL. To dissect mechanisms behind these relationships, we scored sex-specific floral visitation, and the contributions of each pollinator to plant fitness using single flower visits. Small bees and M. campanulae overvisited male-phase flowers, but bumblebees frequently visited female-phase flowers. Fewer bumblebee visits were required to saturate seed set compared to other bees. Scaling pollinator efficiency metrics to populations, small bees deplete large amounts of pollen due to highly male-biased flower visitation and infrequent pollen deposition. Thus, small bees reduce plant reproduction by limiting pollen available for transfer by efficient pollinators, and appear to exploit the plant-pollinator mutualism, acting as functional parasites to C. americana It is therefore unlikely that small bees will compensate for reproductive failure in C. americana when bumblebees are scarce.

Geographic variation in pollen color is associated with temperature stress.

The evolution of flower color, especially petal pigmentation, has received substantial attention. Less understood is the evolutionary ecology of pollen pigmentation, though it varies among and within species and its biochemical properties affect pollen viability. We characterize the distribution of pollen color across 24 populations of the North American herb Campanula americana, and assess the degree to which this variation is genetically based. We identify abiotic factors that covary with pollen color and test whether germination of light and dark pollen is differentially affected by variable temperature and UV. Pollen color varies from white to deep purple in C. americana and is genetically determined. There was a longitudinal cline whereby pollen was darkest in western populations. Accounting for latitudinal variation, western populations experience elevated temperature and UV irradiance. Germination of light-colored pollen was reduced by 60% under high temperature, but dark pollen was unaffected. Exposure to UV reduced germination of light and dark pollen similarly. The cline in pollen color across the range may reflect adaptation to heat stress. This study supports thermal tolerance as a novel function of pollen pigmentation and contributes to growing evidence that abiotic factors can drive floral diversity.

Effects of glaciation and whole genome duplication on the distribution of the Campanula rotundifolia polyploid complex.

PREMISE OF THE STUDY: Both intrinsic and extrinsic factors contribute to a species distribution. Among plants, the extrinsic effects of glaciation and intrinsic effects of whole genome duplication are powerful drivers of biogeographical patterns, but the interplay of these factors is poorly understood. Here, we investigate the roles glaciation and whole-genome duplication have played in the evolution of the widespread polyploid complex Campanula rotundifolia. METHODS: We assessed the cytotype of 37 populations that spanned the geographic and cytotypic range of the C. rotundifolia complex. We constructed a chloroplast phylogeny for these populations and used RAD-seq to create nuclear phylogenies and networks for a subset of 23 populations; and estimated divergence times of major clades using Bayesian estimation of substitution rates. KEY RESULTS: Campanula rotundifolia originated in south-central Europe and underwent range expansion throughout much of Europe and North America. Multiple genome duplications have occurred in C. rotundifolia-at least two tetraploid and three hexaploid formations. CONCLUSIONS: Nuclear and chloroplast phylogenies are largely congruent with a history of populations surviving glacial maxima in known Pleistocene refugia in Europe and North America. Divergent European clades are consistent with two disjunct glacial refugia within Europe. North America was colonized by hexaploids derived from Western European lineages. A glacial refugium in Midwestern North America likely facilitated post-glacial recolonization of North America and limited genetic divergence. These results implicate both glaciation and whole-genome duplication as contributing factors to the extant biogeography of C. rotundifolia.

Timing is everything: Dichogamy and pollen germinability underlie variation in autonomous selfing among populations.

PREMISE OF THE STUDY: The evolution of multiple floral traits often underlies the transition from outcrossing to selfing. Such traits can influence the ability to self, and the timing at which selfing occurs, which in turn affects the costs of selfing. Species that display variation in autonomous selfing provide an opportunity to dissect the phenotypic changes that contribute to variability in the mating system. METHODS: In a common garden, we measured dichogamy and herkogamy in 24 populations of the protandrous mixed-mating herb Campanula americana, and related these to autonomous fruit set (autonomy). We then measured the timing of self-pollen deposition and fruit production in populations with high and low autonomy, and determined whether pollen germinability across floral development contributes to variation in autonomy. KEY RESULTS: Populations that transitioned more rapidly to female phase displayed elevated autonomous selfing, but herkogamy was unassociated with autonomous selfing. Selfing occurred more rapidly in highly autonomous populations because of greater self-pollen deposition early in female phase. Pollen germinability in low-autonomy populations remained constant across floral development, but in high-autonomy populations it increased after floral anthesis and was highest near the onset of female phase. CONCLUSIONS: Reduced dichogamy, elevated self-pollen deposition, and higher pollen germination late in male phase contribute to both earlier selfing and greater selfing. These traits vary among populations, likely reflecting past selection on the mating system. While delayed selfing bears fewer fitness costs, the evolution of earlier selfing may be favored if self-pollen availability decreases over floral development.

Intercontinental dispersal and whole-genome duplication contribute to loss of self-incompatibility in a polyploid complex.

PREMISE OF THE STUDY: Angiosperm species often shift from self-incompatibility to self-compatibility following population bottlenecks. Across the range of a species, population bottlenecks may result from multiple factors, each of which may affect the geographic distribution and magnitude of mating-system shifts. We describe how intercontinental dispersal and genome duplication facilitate loss of self-incompatibility. METHODS: Self and outcross pollinations were performed on plants from 24 populations of the Campanula rotundifolia polyploid complex. Populations spanned the geographic distribution and three dominant cytotypes of the species (diploid, tetraploid, hexaploid). KEY RESULTS: Loss of self-incompatibility was associated with both intercontinental dispersal and genome duplication. European plants were largely self-incompatible, whereas North American plants were intermediately to fully self-compatible. Within both European and North American populations, loss of self-incompatibility increased as ploidy increased. Ploidy change and intercontinental dispersal both contributed to loss of self-incompatibility in North America, but range expansion did not affect self-incompatibility within Europe or North America. CONCLUSIONS: When species are subject to population bottlenecks arising through multiple factors, each factor can contribute to self-incompatibility loss. In a widespread polyploid complex, the loss of self-incompatibility can be predicted by the cumulative effects of whole-genome duplication and intercontinental dispersal.

Postzygotic isolation varies by ploidy level within a polyploid complex.

Whole genome duplication is considered to be a significant contributor to angiosperm speciation due to accumulation of rapid, strong interploid reproductive isolation. However, recent work suggests that interploid reproductive isolation may not be complete, especially among higher order cytotypes. This study evaluates postzygotic reproductive isolation among three cytotypes within a polyploid complex. We conducted reciprocal crosses using two diploid and two hexaploid populations each crossed to tetraploid populations spanning the geographic and phylogenetic range of the Campanula rotundifolia polyploid complex. Interploid and intrapopulation crosses were scored for fruit set, seed number, germination proportion and pollen viability. Postzygotic isolation was calculated for each cross as the product of these fitness components. A subset of offspring was cytotyped via flow cytometry. Postzygotic isolation was significantly lower in tetraploid-hexaploid crosses than diploid-tetraploid crosses, mostly due to substantially higher germination among tetraploid-hexaploid crosses. Tetraploid-hexaploid crosses produced pentaploids exclusively, whereas diploid-tetraploid crosses produced both triploids and tetraploids in high frequencies. Postzygotic isolation was weaker among higher order polyploids than between diploids and tetraploids, and unreduced gametes may facilitate diploid-tetraploid reproduction. This incomplete postzygotic isolation could allow ongoing interploid gene flow, especially among higher order polyploids, which may slow divergence and speciation in polyploid complexes.

Biparental chloroplast inheritance leads to rescue from cytonuclear incompatibility.

Although organelle inheritance is predominantly maternal across animals and plants, biparental chloroplast inheritance has arisen multiple times in the angiosperms. Biparental inheritance has the potential to impact the evolutionary dynamics of cytonuclear incompatibility, interactions between nuclear and organelle genomes that are proposed to be among the earliest types of genetic incompatibility to arise in speciation. We examine the interplay between biparental inheritance and cytonuclear incompatibility in Campanulastrum americanum, a plant species exhibiting both traits. We first determine patterns of chloroplast inheritance in genetically similar and divergent crosses, and then associate inheritance with hybrid survival across multiple generations. There is substantial biparental inheritance in C. americanum. The frequency of biparental inheritance is greater in divergent crosses and in the presence of cytonuclear incompatibility. Biparental inheritance helps to mitigate cytonuclear incompatibility, leading to increased fitness of F1 hybrids and recovery in the F2 generation. This study demonstrates the potential for biparental chloroplast inheritance to rescue cytonuclear compatibility, reducing cytonuclear incompatibility's contribution to reproductive isolation and potentially slowing speciation. The efficacy of rescue depended upon the strength of incompatibility, with a greater persistence of weak incompatibilities in later generations. These findings suggest that incompatible plastids may lead to selection for biparental inheritance.

A geographic cline in the ability to self-fertilize is unrelated to the pollination environment.

The reproductive assurance (RA) hypothesis predicts that the ability to autonomously self-fertilize should be favored in environments where a lack of mates or pollinators limits outcross reproduction. Because such limits to outcrossing are predicted to be most severe at range edges, elevated autonomy in peripheral populations is often attributed to RA. We test this hypothesis in 24 populations spanning the range of Campanula americana, including sampling at the range interior and three geographic range edges. We scored autonomous fruit set in a pollinator-free environment and detected clinal variation-autonomy increased linearly from the southern to the northern edge, and from the eastern to the western edge. We then address whether the cline reflects the contemporary pollination environment. We measured population size, plant density, pollinator visitation, outcross pollen limitation and RA in natural populations over two years. Most populations were pollen limited, and those that experienced higher visitation rates by bumblebees had reduced pollen limitation. Reproductive assurance, however, was generally low across populations and was unrelated to pollen limitation or autonomy. Neither pollen limitation nor RA displayed geographic clines. Finally, autonomy was not associated with pollinator visitation rates or mate availability. Thus, the data do not support the RA hypothesis; clinal variation in autonomy is unrelated to the current pollination environment. Therefore, geographic patterns of autonomy are likely the result of historical processes rather than contemporary natural selection for RA.

Evolution of marginal populations of an invasive vine increases the likelihood of future spread.

The prediction of invasion patterns may require an understanding of intraspecific differentiation in invasive species and its interaction with climate change. We compare Japanese honeysuckle (Lonicera japonica) plants from the core (100-150 yr old) and northern margin (< 65 yr old) of their North American invaded range to determine whether evolution during invasion increases the probability of future expansion. Plants from populations in the core and margin were compared in two sites beyond the northern range edge to assess their potential to invade novel areas. Data were compared with previous work to assess the effect of latitudinal climate on L. japonica spread. Winter survival in current climates was modeled and projected for future climates to predict future spread. Margin plants were larger and had 60% greater survival than core plants at sites beyond the northern range edge. Overall, winter survival decreased with increasing latitude and decreasing temperature, and was greater in margin plants than core plants. Models suggested that greater winter tolerance in margin populations has increased L. japonica's northward spread by 76 km, and that this survival advantage will persist under future climates. These results demonstrate that evolution during invasion may increase spread beyond predictions using increasing global temperatures alone.

The mean and variability of a floral trait have opposing effects on fitness traits.

BACKGROUND AND AIMS: Floral traits are essential for ensuring successful pollination and reproduction in flowering plants. In particular, style and anther positions are key for pollination accuracy and efficiency. Variation in these traits among individuals has been well studied, but less is known about variation within flowers and plants and its effect on pollination and reproductive success. METHODS: Style deflexion is responsible for herkogamy and important for pollen deposition in Passiflora incarnata. The degree of deflexion may vary among stigmas within flowers as well as among flowers. We measured the variability of style deflexion at both the flower and the plant level. The fitness consequences of the mean and variation of style deflexion were then evaluated under natural pollination by determining their relationship to pollen deposition, seed production and average seed weight using structural equation modelling. In addition, the relationship between style deflexion and self-pollen deposition was estimated in a greenhouse experiment. KEY RESULTS: We found greater variation in style deflexion within flowers and plants than among plants. Variation of style deflexion at the flower and plant level was positively correlated, suggesting that variability in style deflexion may be a distinct trait in P. incarnata. Lower deflexion and reduced variation in that deflexion increased pollen deposition, which in turn increased seed number. However, lower styles also increased self-pollen deposition. In contrast, higher deflexion and greater variability of that deflexion increased variation in pollen deposition, which resulted in heavier seeds. CONCLUSIONS: Variability of style deflexion and therefore stigma placement, independent from the mean, appears to be a property of individual P. incarnata plants. The mean and variability of style deflexion in P. incarnata affected seed number and seed weight in contrasting ways, through the quantity and potentially quality of pollen deposition. This antagonistic selection via different fitness components may maintain diverse style phenotypes.

Spatiotemporal variation in deer browse and tolerance in a woodland herb.

Herbivory can shape the dynamics of plant populations, including effects on survival and reproduction, and is in turn affected by environmental factors that vary in space and time. White-tailed deer are significant herbivores in North America that have been broadly documented to affect plant reproductive success. If variation in the frequency and impact of herbivory by deer correlates with a broad-scale latitudinal gradient, climactic effects may be important for shaping plant-herbivore interactions, Alternatively, a lack of broad-scale gradients would suggest local factors such as plant community composition and deer densities are affecting herbivory. To investigate broad-scale patterns of deer herbivory, we examined the frequency and reproductive consequences of deer browse over three years in 17 populations of Campanulastrum americanum spanning the latitudinal extent of its range. Even though deer are overabundant throughout the range of C. americanum, we found spatiotemporal variation in deer browse frequency (0-0.96, mean 0.46) and its effects on plant reproductive success. The four southernmost populations experienced high levels of herbivory, and were responsible for generating a negative relationship between latitude and herbivory. In general, patterns of variation in the frequency and impact of herbivory across the entire latitudinal gradient pointed to the importance of local rather than broad-scale factors. Within a population, deer consumed larger plants. Across many populations and years, average fitnesses of browsed and uneaten plants were similar, suggesting that plants are tolerant to browse. However, since large plants have greater reproductive success and are more likely to be browsed, tolerance may be influenced by plant size. When plant size was accounted for, most populations did not fully compensate for browsing. There was no relationship between browsing intensity and tolerance, suggesting that browsing may be too variable to consistently select for tolerance, or that increases in deer density are too recent for increased tolerance to evolve.

Multiple glacial refugia lead to genetic structuring and the potential for reproductive isolation in a herbaceous plant.

PREMISE OF THE STUDY: Glacial cycles have influenced the genetic structure of many species. In addition to facilitating genetic divergence, isolation in multiple glacial refugia may have contributed to the development of genetic incompatibility and reproductive isolation. We examined the phylogeography of Campanulastrum americanum, a monocarpic herbaceous plant that exhibits strong intraspecific reproductive isolation, to determine whether the current genetic structure reflects a history of multiple glacial refugia. METHODS: Chloroplast loci and nuclear RAD sequencing were used to characterize the range-wide phylogeography of C. americanum, in order to determine locations of potential glacial refugia and recolonization routes. Potential locations of refugia during the Last Glacial Maximum were also identified using ecological niche modeling. KEY RESULTS: Together, the chloroplast and nuclear phylogenies found support for three geographically structured, genetically divergent lineages, among which gene flow appears to be restricted. The distribution of these lineages indicates that C. americanum survived the Last Glacial Maximum in at least three refugia located in the Appalachians and on the Atlantic and Gulf coasts. The ecological niche model also supported the existence of multiple refugia. CONCLUSIONS: The isolation of populations of C. americanum in multiple refugia has led to a degree of phylogeographic structure greater than that found in most previously studied plants in eastern North America, which may be attributable to its short generation time. Reproductively isolated populations of C. americanum belong to divergent lineages, which suggests that survival in multiple glacial refugia contributed to the development of reproductive isolation in this species.

Correlation between sequence divergence and polymorphism reveals similar evolutionary mechanisms acting across multiple timescales in a rapidly evolving plastid genome.

BACKGROUND: Although the plastid genome is highly conserved across most angiosperms, multiple lineages have increased rates of structural rearrangement and nucleotide substitution. These lineages exhibit an excess of nonsynonymous substitutions (i.e., elevated dN/dS ratios) in similar subsets of plastid genes, suggesting that similar mechanisms may be leading to relaxed and/or positive selection on these genes. However, little is known regarding whether these mechanisms continue to shape sequence diversity at the intraspecific level. RESULTS: We examined patterns of interspecific divergence and intraspecific polymorphism in the plastid genome of Campanulastrum americanum, and across plastid genes found a significant correlation between dN/dS and pN/pS (i.e., the within-species equivalent of dN/dS). A number of genes including ycf1, ycf2, clpP, and ribosomal protein genes exhibited high dN/dS ratios. McDonald-Kreitman tests detected little evidence for positive selection acting on these genes, likely due to the presence of substantial intraspecific divergence. CONCLUSIONS: These results suggest that mechanisms leading to increased nucleotide substitution rates in the plastid genome are continuing to act at the intraspecific level. Accelerated plastid genome evolution may increase the likelihood of intraspecific cytonuclear genetic incompatibilities, and thereby contribute to the early stages of the speciation process.

Clinal variation for only some phenological traits across a species range.

Phenology is the timing of life cycle events of an organism. Alterations in phenology can have profound effects on individual fitness, population growth, and community dynamics. Recent changes in climate have altered the phenology of many organisms, which may result in selection to shift phenological traits. Understanding the relationship between local climates and population differentiation in phenology will allow us to anticipate responses to novel selective environments caused by global climate change. We evaluated population differentiation in the number of days to germination, first flower, and fruit maturation for 33 populations throughout the range of Campanulastrum americanum (American Bellflower). Germination and fruit maturation had geographical clines with earlier timing in populations from northern latitudes. Northern sites were cooler and drier, suggesting potential adaptive differentiation of the shorter life cycle associated with earlier phenology. Similarly, higher elevations were cooler and had earlier fruit maturation. However, seed germination was later in higher elevation populations. Although there was substantial variation in the day to first flower, ranging 40 days between population means, it was idiosyncratic and not related to latitude, suggesting differentiation in response to selective factors distinct from those on germination and fruit maturation. Thus, germination and fruit maturation in C. americanum may shift in response to selection by rising temperatures. However, such changes are not expected for flowering time, a typical indicator of climate change.