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.

Variation in heteroploid reproduction and gene flow across a polyploid complex: One size does not fit all.

Whole-genome duplication is considered an important speciation mechanism in plants. However, its effect on reproductive isolation between higher cytotypes is not well understood. We used backcrosses between different ploidy levels and surveys of mixed-ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. We backcrossed F1 hybrids derived from 2X-4X and 4X-6X crosses in the Campanula rotundifolia autopolyploid complex, measured backcross fitness, and estimated backcross DNA cytotype. We then sampled four natural mixed-ploidy contact zones (two 2X-4X and two 4X-6X), estimated ploidy, and genotyped individuals across each contact zone. Reproductive success and capacity for gene flow was markedly lower for 2X-4X than 4X-6X hybrids. In fact, 3X hybrids could not backcross; all 2X-4X backcross progeny resulted from neotetraploid F1 hybrids. Further, no 3X individuals were found in 2X-4X contact zones, and 2X and 4X individuals were genetically distinct. By contrast, backcrosses of 5X hybrids were relatively successful, particularly when crossed to 6X individuals. In 4X-6X contact zones, 5X individuals and aneuploids were common and all cytotypes were largely genetically similar and spatially intermixed. Taken together, these results provide strong evidence that reproduction is low between 2X and 4X cytotypes, primarily occurring via unreduced gamete production, but that reproduction and gene flow are ongoing between 4X and 6X cytotypes. Further, it suggests whole-genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes.

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.

Strength in numbers? Cytotype frequency mediates effect of reproductive barriers in mixed-ploidy arrays.

When differentiated lineages come into contact, their fates depend on demographic and reproductive factors. These factors have been well-studied in taxa of the same ploidy, but less is known about sympatric lineages that differ in ploidy, particularly with respect to demographic factors. We assessed prezygotic, postzygotic, and total reproductive isolation in naturally pollinated arrays of diploid-tetraploid and tetraploid-hexaploid population mixes of Campanula rotundifolia by measuring pollinator transitions, seed yield, germination rate, and proportion of hybrid offspring. Four frequencies of each cytotype were tested, and pollinators consistently overvisited rare cytotypes. Seed yield and F1 hybrid production were greater in 4X-6X arrays than 2X-4X arrays, whereas germination rates were similar, creating two distinct patterns of reproductive isolation. In 2X-4X arrays, postzygotic isolation was near complete (3% hybrid offspring), and prezygotic isolation associated with pollinator preference is expected to facilitate the persistence of minority cytotypes. However, in 4X-6X arrays where postzygotic isolation permitted hybrid formation (44% hybrids), pollinator behavior drove patterns of reproductive isolation, with rare cytotypes being more isolated and greater gene flow expected from rare into common cytotypes. In polyploid complexes, both the specific cytotypes in contact and local cytotype frequency, likely reflecting spatial demography, will influence likelihood of gene exchange.

Geographic Variation in Floral Color and Reflectance Correlates With Temperature and Colonization History.

Petal color variation within species is common and may be molded by abiotic or biotic selection pressures, or neutral population structure. For example, darker flowers may be favored in cooler environments because they absorb more solar radiation, elevating the temperature of reproductive structures. Additionally, flower color may evolve to attract the dominant or most efficient pollinator type in a given population. Here, we evaluate geographic variation in petal coloration across the range of Campanula americana in Eastern North America and test whether color covaries with abiotic factors, the pollination community, and genetic structure established through post-glacial expansion. Consistent with other studies, flowers from cooler, higher latitude populations were less reflective across the UV-NIR spectrum than those from warmer populations. Local temperature explained variation in petal reflectance better than the pollinator community or colonization history. Petal color perceived by trichromatic bee pollinators displayed a strong longitudinal pattern but was unassociated with climatic factors and the pollinator community. Instead, pollinator-perceived color was tightly correlated with the geographic distance from C. americana's glacial refugium. In total, abiotic conditions appear to shape large-scale geographic variation in the intensity of petal reflectance while genetic structure is the strongest driver of pollinator-perceived petal coloration. This study highlights the importance of abiotic factors and historical processes associated with range expansion as major evolutionary forces shaping diversity of flower coloration on large geographic scales.

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.

Selfing ability and drift load evolve with range expansion.

Colonization at expanding range edges often involves few founders, reducing effective population size. This process can promote the evolution of self-fertilization, but implicating historical processes as drivers of trait evolution is often difficult and requires an explicit model of biogeographic history. In plants, contemporary limits to outcrossing are often invoked as evolutionary drivers of self-fertilization, but historical expansions may shape mating system diversity, with leading-edge populations evolving elevated selfing ability. In a widespread plant, Campanula americana, we identified a glacial refugium in the southern Appalachian Mountains from spatial patterns of genetic drift among 24 populations. Populations farther from this refugium have smaller effective sizes and fewer rare alleles. They also displayed elevated heterosis in among-population crosses, reflecting the accumulation of deleterious mutations during range expansion. Although populations with elevated heterosis had reduced segregating mutation load, the magnitude of inbreeding depression lacked geographic pattern. The ability to self-fertilize was strongly positively correlated with the distance from the refugium and mutation accumulation-a pattern that contrasts sharply with contemporary mate and pollinator limitation. In this and other species, diversity in sexual systems may reflect the legacy of evolution in small, colonizing populations, with little or no relation to the ecology of modern 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.

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.

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.

Response to joint selection on germination and flowering phenology depends on the direction of selection.

Flowering and germination time are components of phenology, a complex phenotype that incorporates a number of traits. In natural populations, selection is likely to occur on multiple components of phenology at once. However, we have little knowledge of how joint selection on several phenological traits influences evolutionary response. We conducted one generation of artificial selection for all combinations of early and late germination and flowering on replicated lines within two independent base populations in the herb Campanula americana. We then measured response to selection and realized heritability for each trait. Response to selection and heritability were greater for flowering time than germination time, indicating greater evolutionary potential of this trait. Selection for earlier phenology, both flowering and germination, did not depend on the direction of selection on the other trait, whereas response to selection to delay germination and flowering was greater when selection on the other trait was in the opposite direction (e.g., early germination and late flowering), indicating a negative genetic correlation between the traits. Therefore, the extent to which correlations shaped response to selection depended on the direction of selection. Furthermore, the genetic correlation between timing of germination and flowering varies across the trait distributions. The negative correlation between germination and flowering time found when selecting for delayed phenology follows theoretical predictions of constraint for traits that jointly determine life history schedule. In contrast, the lack of constraint found when selecting for an accelerated phenology suggests a reduction of the covariance due to strong selection favoring earlier flowering and a shorter life cycle. This genetic architecture, in turn, will facilitate further evolution of the early phenology often favored in warm climates.

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.

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.

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.

Variation in reproductive isolation across a species range.

Reproductive isolation is often variable within species, a phenomenon that while largely ignored by speciation studies, can be leveraged to gain insight into the potential mechanisms driving the evolution of genetic incompatibilities. We used experimental greenhouse crosses to characterize patterns of reproductive isolation among three divergent genetic lineages of Campanulastrum americanum that occur in close geographic proximity in the Appalachian Mountains. Substantial, asymmetrical reproductive isolation for survival due to cytonuclear incompatibility was found among the lineages (up to 94% reduction). Moderate reductions in pollen viability, as well as cytoplasmic male sterility, were also found between some Mountain populations. We then compared these results to previously established patterns of reproductive isolation between these Mountain lineages and a fourth, widespread Western lineage to fully characterize reproductive isolation across the complete geographic and genetic range of C. americanum. Reproductive isolation for survival and pollen viability was consistent across studies, indicating the evolution of the underlying genetic incompatibilities is primarily determined by intrinsic factors. In contrast, reproductive isolation for germination was only found when crossing Mountain populations with the Western lineage, suggesting the underlying genetic incompatibility is likely influenced by environmental or demographic differences between the two lineages. Cytoplasmic male sterility was also limited in occurrence, being restricted to a handful of Mountain populations in a narrow geographic range. These findings illustrate the complexity of speciation by demonstrating multiple, independent genetic incompatibilities that lead to a mosaic of genetic divergence and reproductive isolation across a species range.

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.

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.

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.