Variation in frequency of hybrids and spatial structure among Ipomopsis (Polemoniaceae) contact sites

Rates of hybridization vary among angiosperm taxa. Among-taxon variation in hybridization rate has been used to compare the importance of pre- and post-zygotic reproductive isolating mechanisms. Variation in rates of hybridization within a single-species pair would suggest that local conditions also affect reproductive isolation within a single taxonomic context. In this study, contact sites of Ipomopsis aggregata-Ipomopsis tenuituba were surveyed for variation in frequency of hybrids, and spatial structure. Floral morphology was used to identify parent species and hybrids in seven contact sites in the western Rocky Mountains, USA. Contact sites varied widely in elevational range, the degree to which morphological variation was clinal rather than mosaic and the frequency of hybrids. Two sites provided a strong contrast between a clinal, unimodal site and a mosaic, bimodal site. This natural variation among contact sites of the same species pair provides an opportunity to assess the effect of local ecological conditions and spatial structure of parent populations on reproductive isolation, while controlling for between-taxon variation.     

Leaf physiology reflects environmental differences and cytoplasmic background in Ipomopsis (Polemoniaceae) hybrids

Natural hybridization can produce individuals that vary widely in fitness, depending upon the performance of particular genotypes in a given environment. In a hybrid zone with habitat heterogeneity, differences in physiological responses to abiotic conditions could influence the fitness and spatial distribution of hybrids and parental species. This study compared gas exchange physiology of Ipomopsis aggregata, I. tenuituba, and their natural hybrids in situ and assessed whether physiological differences were consistent with their native environmental conditions. We also produced reciprocal F2s in a greenhouse study to test for cytonuclear effects on water-use efficiency (WUE). The relative performance of natural hybrids and parentals was consistent with their native habitats: I. aggregata at the coolest, wettest locations had the lowest WUE, while hybrids from the most xeric sites had the highest WUE. In hybrids, the mechanism by which both natural and experimental hybrids achieved this high WUE depended on cytotype: those with I. tenuituba cytoplasm had reduced transpiration, while those with I. aggregata cytoplasm had an increased photosynthetic rate, consistent with patterns in the cytoplasmic parent. The high WUE in hybrids may contribute to their high survival in the dry center of the natural hybrid zone, consistent with environment-dependent models of hybrid zone dynamics.     

Floral scent in natural hybrids of Ipomopsis (Polemoniaceae) and their parental species

Background and Aims

Floral traits, such as floral volatiles, can contribute to pre-zygotic reproductive isolation by promoting species-specific pollinator foraging. When hybrid zones form, floral traits could also influence post-zygotic isolation. This study examined floral volatiles in parental species and natural hybrids in order to explore potential scent mediation of pre-zygotic and post-zygotic isolation.

Methods

Floral bouquets were analysed for the sister species Ipomopsis aggregata and I. tenuituba and their natural hybrids at two contact sites differing in both hybridization rate and temporal foraging pattern of hawkmoth pollinators. Floral volatiles were quantified in diurnal and nocturnal scent samples using gas chromatography–mass spectrometry.

Key Results

The bouquets of parental species and hybrids showed qualitative overlap. All flowers emitted similar sets of monoterpenoid, sesquiterpenoid, aliphatic and benzenoid compounds, but separated into groups defined by multivariate analysis of quantitative emissions. The parental species differed most strikingly in the nitrogenous compound indole, which was found almost exclusively in nocturnal bouquets of I. tenuituba. Natural hybrid bouquets were highly variable, and showed emission rates of several compounds that appeared transgressive. However, indole emission rates were intermediate in the hybrids compared with rates in the parents. Volatile bouquets at the contact site with lower hybridization did not show greater species specificity in overall scent emission, but I. tenuituba presented a stronger indole signal during peak hawkmoth activity at that site.

Conclusions

The two species of Ipomopsis differed in patterns of floral bouquets, with indole emitted in nocturnal I. tenuituba, but not in I. aggregata. Natural hybrid bouquets were not consistently intermediate between the parents, although hybrids were intermediate in indole emission. The indole signal could potentially serve as a hawkmoth attractant that mediates reproductive isolation both before and after hybrid formation.     

Patterns of color and nectar variation across an Ipomopsis (Polemoniaceae) hybrid zone

Hybridization may uncouple adaptive trait combinations that are present in parental species. I studied variation in flower color and reward quality across a hybrid zone of Ipomopsis aggregata and I. tenuituba. Individuals from hybrid populations showed considerable variation in flower color using corolla reflectance measurements. Flower spectra of such individuals were either intermediate or else resembled those flowers from the parental species. Ipomopsis aggregata populations had consistently higher nectar production rates and higher nectar standing crops than either I. tenuituba or hybrids. Ipomopsis aggregata flowers also produced more dilute nectar than those of hybrids and I. tenuituba, but the actual concentration values were quite variable among populations of the same type. Overall, the nectar quality of hybrid flowers most resembled that of I. tenuituba flowers. Based on the observed interpopulation patterns of color-reward associations in this hybrid zone, pollinators should be able to discriminate against I. tenuituba and hybrid populations and against most individuals within hybrid populations. However, they may visit those hybrids that resemble the most rewarding flower type (I. aggregata). The results emphasize the need for studies that address how hybridization affects subsequent plant fitness and the evolutionary dynamics of the species involved.     

Multiple paternity in fruits of Ipomopsis aggregata (Polemoniaceae)

Two different mechanisms can result in multiple paternity within fruits: deposition of a mixed pollen load due to carryover of pollen from flower to flower and multiple pollinator visits in close succession. I investigated the extent of multiple paternity within fruits of Ipompsis aggregata containing from 2 to 14 seeds. A paternity analysis based on ten polymorphic isozyme markers revealed multiple paternity in a minimum of 68% (based on simple paternity exclusion) and up to 100% (based on identification of the most likely father) of the multiseeded fruits. The estimated number of fathers increased with the number of seeds in a fruit, with an average of four sires, and up to nine sires, represented in a single fruit. To explore whether this level of multiple paternity could be explained solely by simultaneous deposition of a mixed pollen load, I constructed a computer simulation model based on previous measurements of movement patterns and pollen carryover by the hummingbird pollinators. Model predictions provided a good match to observed values for number of sires per fruit. Thus, the extensive pollen carryover in this species and consequent mixed pollen loads can explain the high levels of multiple paternity in natural populations.     

Characterization of microsatellite loci in Ipomopsis (Polemoniaceae) wildflowers

Members of the Ipomopsis aggregata species complex in the phlox family (Polemoniaceae) often hybridize when they occur in sympatry, and thus have been extensively studied to examine processes involved in plant speciation. I developed 12 microsatellite loci in I. aggregata that are also polymorphic in closely related Ipomopsis tenuituba, producing an average of eight alleles per locus in test populations of 12 individuals per species. Several of these markers also successfully amplified in other Ipomopsis species and more distant members of the Polemoniaceae, suggesting they should prove useful for a broad range of evolutionary studies in this widely distributed system.     

Asymmetrical pollen success in Ipomopsis (Polemoniaceae) contact sites

Variation in hybridization rates among contact sites of a species pair provides an opportunity for assessing the importance of individual reproductive isolating mechanisms in limiting gene flow between species and thus promoting speciation. Conspecific pollen advantage is common in angiosperms, but its importance as a reproductive isolating mechanism is uncertain. We compared the strength of conspecific pollen advantage in two Ipomopsis aggregata-I. tenuituba (Polemoniaceae) contact sites that differ in frequency of natural hybrids. We performed hand pollinations of single- and 1:1 mixed-species pollen loads, using donor and recipient plants from both contact sites. Paternity of offspring from mixed-species pollinations was determined using an allozyme marker. Donors from the high frequency hybrid site showed no conspecific pollen advantage; both species sired seeds in proportion to their fraction of the pollen load (0.5). In contrast, I. aggregata from the low frequency hybrid site sired 70-85% of offspring on recipients from both sites. These results suggest that pollen interactions can influence the level of natural hybridization. They also suggest the importance of geographic variation in reproductive isolation, which should be considered in studies of biological invasions and exposure of engineered crops to wild relatives.     

Tests of pre- and postpollination barriers to hybridization between sympatric species of Ipomopsis (Polemoniaceae)

The Ipomopsis aggregata species complex (Polemoniaceae) includes species pairs that hybridize readily in nature as well as pairs that meet along contact zones with no apparent hybridization. Artificial hybrids can be made between I. aggregata and I. arizonica, yet morphological intermediates between these two species have not been observed in natural populations. This apparent lack of hybridization is perplexing given that plants of the two species often grow within a few metres of each other and both species have red flowers visited by the same species of hummingbirds. We used trained hummingbirds to examine pollen transfer within and between species. We also hand-pollinated flowers to examine paternal success of heterospecific and conspecific pollen, testing paternity with electrophoretic examination of seeds. Hummingbirds were not simply better at transferring pollen within than between species. Instead, I. arizonica was a better pollen donor so that considerable pollen transfer was observed from I. arizonica to I. aggregata, but very little in the opposite direction. Conversely, once pollen arrived at stigmas, I. arizonica pollen performed very poorly on I. aggregata pistils. However, pollen from I. aggregata could, in some cases, sire seeds on I. arizonica. We hypothesize that hybrids are scarce in nature, in part, because of asymmetric barriers to reproduction: little pollen transfer in one direction and poor pollen performance in the other.     

Investigating species boundaries in the Giliopsis group of Ipomopsis (Polemoniaceae): Strong discordance among molecular and morphological markers

As a first step in elucidating mechanisms of speciation in the Giliopsis group of Ipomopsis (Polemoniaceae), we examined patterns of morphological and genetic differentiation and crossability. This group comprises three species that diverged very recently: two perennials, I. guttata and I. tenuifolia, and one annual, I. effusa. Analysis of phenotypic variation established that the three species are distinct for floral characters, and this differentiation is maintained in a locality containing both perennial species. Next, we assessed the genealogical relationships with AFLPs. All sampled individuals of I. effusa clustered together, a result in accord with its genetic isolation. The perennials, which retain interfertility, were not resolved as sister taxa. Rather, individuals sampled from the single I. guttata population that is sympatric with I. tenuifolia were genetically more similar to I. tenuifolia samples than they were to conspecifics. This pattern may be due to substantial introgression of I. tenuifolia genomic regions that do not contribute to floral phenotype in I. guttata. Our result adds to mounting evidence that plant species, as defined by morphological characters, are often not genomically cohesive. Taken together, our data warrant caution in delimiting species with genetic markers alone, and, importantly, suggest that selection on species-diagnostic morphological characters can be sufficiently strong to counteract extensive gene flow.     

Self-sterility in Ipomopsis aggregata (Polemoniaceae) is due to prezygotic ovule degeneration

Based on previous studies, extreme (>99%) self-sterility in scarlet gilia (Ipomopsis aggregata) appears to be involved in late-acting ovarian self-incompatibility (OSI). Here, we confirm this suggestion by comparing structural events that follow from cross- vs. self-pollinations of I. aggregata. Growth of cross- and self-pollen tubes in the style at 11 h and growth in the ovary at 24 h was equivalent. Nonetheless, by 24 h, cross-pollen effected a significantly higher percentage of both ovule penetration and fertilization. Ovules in self-pollinated flowers showed pronounced changes, including an absence of embryo sac expansion and reduced starch in the integument, by 11 h post-pollination, well before pollen tube entry into the ovary. In addition, the integumentary tapetum and adjacent 1-3 cell layers exhibited abnormal cell division, pronounced deposition of thick, pectin-rich cell walls, and cellular collapse. Ovules and embryo sacs from cross-pollinated flowers rarely showed such features. Developmental changes in ovules from self-pollinated flowers eventually resulted in integument and embryo sac collapse, a process not observed in ovules of unpollinated flowers. We suggest that OSI involves long-distance signaling between self-pollen or self-pollen tubes and carpel tissue that reduces availability of receptive ovules for fertilization before pollen tubes arrive in the ovary.     

Maternal flower color,ultraviolet protection,and germination in Ipomopsis aggregata (Polemoniaceae)

Transgenerational interactions between flower color, seed quality, and seedling performance have rarely been investigated. The ecological model, Ipomopsis aggregata, is a great candidate for examining the maternal effects of flower color because it is a mostly scarlet-flowering plant which shows color polymorphism within natural populations. Anthocyanin, the red flavonoid pigment which gives these flowers color, has been shown to act as an ultraviolet (UV) protectant by shielding chloroplasts and acting as an antioxidant. This study was conducted on scarlet- and fuchsia-flowering maternal plants and their seeds from natural populations in Colorado. Dark-flowering (scarlet) maternal plants from these populations had consistently higher foliar anthocyanin content, photosystem efficiency, and chlorophyll content than light-flowering (fuchsia) plants over a 3-year period in the field. Seeds from a subset of these maternal plants were counted, weighed, and germinated in a growth chamber. Photosystem efficiency, vegetative anthocyanin content, chlorophyll content, and biomass were measured on germinated seedlings after the germination census was completed. Dark-flowering maternal plants yielded seeds and seedlings with higher biomass than light-flowering ones. Seeds from dark-flowering maternal plants also germinated faster than those from light-flowering maternal plants and seedlings had higher vegetative anthocyanin content. The hereditary nature of anthocyanin content thus suggests that higher anthocyanin levels (both floral and vegetative) are potentially linked to measures of fitness such as increased seed weight, germination rate, and seedling biomass. These data suggest that UV protection provided by anthocyanins potentially increases the realized fitness of maternal plants, thereby influencing life history.     

Frequency of spontaneous amphiploids in Gilia (Polemoniaceae) hybrids

This paper reports the frequency of spontaneous amphiploids or neopolyploids in experimental interspecific hybrids in two annual sections of Gilia (Polemoniaceae, sects. Arachnion and Gilia, x = 9). In each section the species were intercrossed in numerous hybrid combinations to obtain an array of F1 hybrid types; seven of the hybrid types spontaneously produced new amphiploid progeny in F2. In sect. Arachnion the parental species and F1s were all diploids and the amphiploids were tetraploid; in sect. Gilia the parental species were diploids or tetraploids and the amphiploids were tetraploid or hexaploid. Two measures were used to express the frequency of spontaneous amphiploid formation. The first is the proportion of the F1 hybrid combinations that yielded any amphiploid progeny at all. In sect. Arachnion this proportion is 3/39 or 7.7%; in sect. Gilia it is 4/16 or 25%. A second measure is needed to express the variation in productivity among the seven amphiploid-producing hybrid types. The measure adopted is the mean number of new amphiploids per single F1 hybrid plant. The productivity numbers for the three hybrid combinations in sect. Arachnion are 0.25, 0.67, and 2.0; and those for four hybrid combinations in sect. Gilia are 0.15, 0.62, 2.25, and 12.62. There are large differences from one hybrid combination to another in rate of amphiploid formation, and the rate is quite high in some hybrids. The factors affecting these differences are discussed.     

Absence of conspecific pollen advantage in the dynamics of an Ipomopsis (Polemoniaceae) hybrid zone

The frequency of hybrid formation in angiosperms depends on how often heterospecific pollen is transferred to the stigma and on the success of that heterospecific pollen at fertilizing ovules. Even if heterospecific pollen is capable of effecting fertilization it may perform poorly when conspecific pollen is also available on the stigma. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Ipomopsis (Polemoniaceae) in Colorado. Plants of both parental species and natural hybrids were pollinated with I. aggregata and I. tenuituba pollen in ratios of 100:0, 80:20, 50:50, 20:80, and 0:100 by mass. Plants were homozygous for different alleles at an isozyme marker, allowing us to distinguish the type of pollen parent for 2166 viable seeds from 273 fruits. In contrast to studies of many other hybridizing taxa, there was no evidence of an advantage to conspecific pollen, nor did composition of the stigmatic pollen load affect seed set. Instead, the frequency of seeds sired by a given species was proportional to its representation in the pollen load. In this hybrid zone, both the frequency of first-generation hybrid formation and the relative male fitness of the two parental species should be predictable from the rates of pollen transfer to stigmas.     

Species-independent, geographical structuring of chloroplast DNA haplotypes in a montane herb Ipomopsis (Polemoniaceae)

Two hypotheses have been proposed to explain the occurrence of hybrid zones between red-flowered Ipomopsis aggregata and white-flowered I. tenuituba . Either local adaptation to hummingbird and hawkmoth pollinators has given rise to sympatric (or parapatric) divergence of flower colour and morphology (primary intergradation at hybrid zones), or alternatively two previously allopatric species are coming into contact at several geographical areas of secondary intergradation. We examined restriction site patterns in nuclear DNA (nrDNA), chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) from populations of I. aggregata and I. tenuituba representing seven zones of sympatry. No variation was detected in a 350-bp fragment of mtDNA and uninformative levels of variation were observed for nrDNA. We detected 22 potentially informative restriction site polymorphisms in cpDNA, all of which united geographical areas containing populations of both species. We detected no informative species-specific markers. Studies of other species (e.g. oaks) have detected similar species-independent geographical structure of cpDNA. However, in these cases secondary interegradation could be inferred from species-specific nuclear alleles. The pattern in Ipomopsis is consistent with both primary intergradation (independent speciation in each area of sympatry) or secondary intergradation involving complete cytoplasmic replacement. Thus, additional data are needed to explain the origin of hybrid zones in Ipomopsis .     

Pollination of Ipomopsis aggregata (Polemoniaceae): effects of intra- vs. interspecific competition

Although plants may simultaneously experience intra- and interspecific competition for pollination, their relative strength has rarely been experimentally evaluated. Yet because intra- and interspecific competition can be caused by different mechanisms, their effect on the ecology and evolution of plants may differ. To determine the relative strength of intra- and interspecific competition for pollination, I manipulated the presence of heterospecifics and density of conspecifics using Ipomopsis aggregata as the focal species. All plots contained I. aggregata and Castilleja linariaefolia, but C. linariaefolia inflorescences were removed from half of the plots to create the heterospecifics-absent treatment. Within each plot, all I. aggregata inflorescences were removed from a 5-m radius around a focal plant to create a low conspecific density experimental unit, and a group of 12 I. aggregata plants/1 m² was designated as a high conspecific density unit. Conspecific pollen deposition was reduced when C. linariaefolia was present but was not influenced by I. aggregata density. Although seed set per fruit was reduced by 17% when C. linariaefolia was present, it was not significantly influenced by either treatment. Interspecific competition for pollination is stronger than intraspecific competition in the I. aggregata–C. linariaefolia system, but neither process appears to influence plant fitness.     

Characterizing Ipomopsis rubra (Polemoniaceae) germination under various thermal scenarios with non-parametric and semi-parametric statistical methods

Time-to-event analysis represents a collection of relatively new, flexible, and robust statistical techniques for investigating the incidence and timing of transitions from one discrete condition to another. Plant biology is replete with examples of such transitions occurring from the cellular to population levels. However, application of these statistical methods has been rare in botanical research. Here, we demonstrate the use of non- and semi-parametric time-to-event and categorical data analyses to address questions regarding seed to seedling transitions of Ipomopsis rubra propagules exposed to various doses of constant or simulated seasonal diel temperatures. Seeds were capable of germinating rapidly to >90 % at 15–25 or 22/11–29/19 °C. Optimum temperatures for germination occurred at 25 or 29/19 °C. Germination was inhibited and seed viability decreased at temperatures ≥30 or 33/24 °C. Kaplan–Meier estimates of survivor functions indicated highly significant differences in temporal germination patterns for seeds exposed to fluctuating or constant temperatures. Extended Cox regression models specified an inverse relationship between temperature and the hazard of germination. Moreover, temperature and the temperature × day interaction had significant effects on germination response. Comparisons to reference temperatures and linear contrasts suggest that summer temperatures (33/24 °C) play a significant role in differential germination responses. Similarly, simple and complex comparisons revealed that the effects of elevated temperatures predominate in terms of components of seed viability. In summary, the application of non- and semi-parametric analyses provides appropriate, powerful data analysis procedures to address various topics in seed biology and more widespread use is encouraged.     

缺齿蓑藓(Macromitrium gymnostomum)的形态和遗传变异及其地理因素分析

缺齿蓑藓(Macromitrium gymnostomum Sull.Lesq.)广布于我国东南部,形态变异较大,与近缘种关系模糊。为了正确鉴定缺齿蓑藓形态变异范围,以及形态、遗传和地理因素三者的关系,对缺齿蓑藓11个地理居群106个样本配子体的13个形态性状进行了测定。结果显示,根据形态数据可将106份样本聚类成8个形态组。用13对ISSR引物获得了150个位点,其中148个为多态位点,多态位点百分率为98.67%; Nei's基因多样性在居群内占62.22%,在居群间占37.78%;居群间基因流(Nm)为0.8235,遗传分化指数(Gst)为0.3778。11个地理居群的遗传距离在0.0873～0.2363之间,平均为0.1508。基于148个多态位点可将106份样本聚成8个遗传组。缺齿蓑藓的形态变异有一定的遗传背景(r=0.159,n=106,P 0.2),地理因素对形态(r=0.309,n=106,P 0.01)和遗传(r=0.251,n=106,P 0.01)分化产生了极显著影响。     

Geographical patterns of morphological variation and environmental correlates in contact zones: a multi-scale approach using two Mediterranean vipers (Serpentes)

Adaptation to similar selective pressures can explain morphological convergence between closely related species in contact zones. Geostatistics and Geographical Information System were used to identify multi-scale patterns of morphological variability and test the hypothesis of morphological convergence due to local environmental pressures in the contact areas between Vipera aspis and Vipera latastei in the Iberian Peninsula. Nine morphological traits from 630 and 362 vipers at regional and local scale, respectively, were interpolated by Kriging to generate surfaces of morphological variation. Kriging is a geostatistical algorithm that allows investigating the spatial structure of data with statistical models. At both scales, a convergent north–south pattern in morphological variability was observed and the contact areas were identified as integration zones where intermediate vipers are found. Significant correlations were found between surfaces of univariate and multivariate traits, with precipitation and temperature seasonality. Thus, several morphological traits were apparently under local environmental selection. Nevertheless, the influence of biotic pressures and gene flow on morphological convergence of vipers in contact zones deserves further study.     

Natural hybridization between an outcrossing and a selfingPhlox (Polemoniaceae): The maternal species of F1 hybrids

Natural F₁ hybrids between the outcrossingPhlox drummondii and the predominantly selfingP. cuspidata were examined to ascertain the proportion of hybrid individuals mothered by each species. Species-specific restriction fragment patterns (both nrDNA and cpDNA) were established as markers, and synthetic hybrids of known parentage were utilized to determine that the chloroplast genome is maternally inherited. Of 89 mature natural hybrids examined, approximately two thirds were mothered byP. drummondii, the outcrosser. That the outcrosser should mother most hybrids is expected since it is dependent upon incoming pollen for fertilization, and hybrids may result when heterospecific pollen is received. The fact that the highly selfingP. cuspidata mothered nearly one third of the hybrids is surprising, and may be related to both pre-zygotic and post-zygotic factors. Which species mothers hybrids has important implications for the potential for introgression as well as its direction.