Post-glacial history of Trillium grandiflorum (Melanthiaceae) in eastern North America: inferences from phylogeography

Dispersal and migration are important processes affecting the evolutionary history and genetics of species. Here we investigate post-glacial migration and gene flow in Trillium grandiflorum (Melanthiaceae), a wide-ranging, forest herb from eastern North America. Using phylogeographic approaches, we examined cpDNA and allozyme diversity in 35 populations of T. grandiflorum sampled from throughout the geographic range of the species. Nested clade analysis (NCA) of cpDNA haplotypes indicated that T. grandiflorum likely survived in two refugia in the southeastern US during the last glaciation and that long-distance dispersal characterized the post-glacial recolonization of northern areas. There was no evidence for reduced allozyme diversity in populations from glaciated compared to ice-free regions, probably because of the greater abundance and larger effective size of populations in the north. An analysis of isolation-by-distance based on the allozyme data suggested a pattern of population differentiation consistent with restricted gene flow. Notwithstanding the significance of rare seed dispersal events for migration, a comparison of allozyme and cpDNA genetic structure indicates that pollen flow between populations is more likely than seed dispersal. These results for T. grandiflorum represent the first phylogeographic analysis of a temperate woodland herb in eastern North America and support the importance of occasional long-distance dispersal events in the post-glacial migration of plants.     

Factors affecting the initiation of mini-rhizomes from Trillium erectum and T. grandiflorum tissues in vitro

Leaf and stem explants of Trillium grandiflorum and T. erectum produced mini-rhizomes (MRs) in vitro which gave rise to shoots and roots. The apical portion of the stem and the basal portion of the leaves were the most effective explants from these tissues, while stem tissue was more responsive than leaf tissue. The best response with both species was observed on half-strength MS basal medium supplemented with cytokinin and auxin. T. erectum was more responsive than T. grandiflorum overall, and in some cases produced MRs in the absence of growth regulators. Culture at 21°C appeared to stimulate growth from T. grandiflorum tissues, compared with controls at 27°C, whereas the outgrowth of shoots from MRs was inhibited in both species at 21°C. In vitro production of MRs could provide a more rapid, alternative propagation method for these species than traditional methods.     

Adaptive significance of self-fertilization in a hermaphroditic perennial, Trillium camschatcense (Melanthiaceae)

The evolution of self-fertilization from primarily outcrossing ancestors is one of the most common evolutionary transitions in plants; however, the ecological mechanisms that maintain self-fertilization have remained controversial. Theoretical studies suggest that selfing is advantageous over outcrossing in terms of genetic transmission and assurance of seed production under pollen-limited circumstances. Trillium camschatcense is a herbaceous perennial distributed in Hokkaido and northern Honshu, Japan. Geographical variation in the breeding system (self-compatible, SC; or self-incompatible, SI) has been reported in populations in Hokkaido. Here, we used several SC and SI populations of T. camschatcense to investigate the adaptive significance and the evolutionary basis of self-fertilization. Pollination experiments and genetic analyses demonstrated that the potential availability of outcross pollen in SC populations was sufficient and that the number of pollen donors was equal to that of SI populations. However, despite the high availability of outcross pollen, the SC populations produced seeds predominantly by selfing and so underwent severe inbreeding depression. Although none of the suggested advantages for self-fertilization were supported by our analyses, we propose two possible scenarios for the evolution of self-fertilization in T. camschatcense.     

Electron microscope autoradiographic studies of RNA metabolism in Trillium erectum microspores

3H-Uridine has been used to investigate the sites of RNA synthesis in the post-meiotic G₁ phase of Trillium microspores using electron microscope autoradiography. The dilute, non-condensed component of the nucleus has been found to be the site of synthesis. When the labelled cells were further incubated in non-radioactive medium the label was found to shift towards the condensed chromatin regions within the nucleus. Two hypotheses to explain the observations are considered, one involving migration of the RNA from the relaxed to condensed regions, the other involving a change in state of the nuclear regions involved in the synthesis. The data are interpreted as favoring the latter possibility.This study supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research and from the Swiss National Fund, Grant Nr. 3202.Fellow of the Jane Coffin Childs Fund for Medical Research.     

Effects of herbivory and its timing across populations of Trillium grandiflorum (Liliaceae)

The goal of this study was to identify the degree to which the frequency and timing of herbivory by white-tailed deer (Odocoileus virginianus) and subsequent plant response varied across 12 populations of the perennial herb Trillium grandiflorum. Effects of natural and experimental herbivory on the stage and size of reproductive plants were measured. Both the frequency and timing of herbivory varied across T. grandiflorum populations. Reproductive plants were more likely to regress to nonreproductive stages in the next growing season when (1) reproductive plants were consumed by deer (vs. intact reproductive plants); (2) reproductive plants were consumed early in the growing season (vs. reproductive plants consumed late in the growing season); (3) reproductive plants were smaller in size. Clipped plants that remained reproductive were smaller in the following season than unclipped controls. Plant size was positively correlated with the number of ovules, suggesting that reductions in the growth rate of reproductive plants diminish their future reproductive success. Populations with high levels of natural herbivory had a greater proportion of reproductive plants that regressed to nonreproductive stages, probably because reproductive plants in these populations were smaller in size. However, the plant response to herbivory was similar across populations.     

Steroidal glycosides from the underground parts of Trillium erectum and their cytotoxic activity

Six steroidal glycosides, along with 14 known compounds, were isolated from the underground parts of Trillium erectum L. (Liliaceae). The structures of 1-6 were determined on the basis of extensive spectroscopic analysis, including two-dimensional (2D) NMR data, and a few chemical transformations. The isolated compounds were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells.     

Impact of tree leaf phenology on growth rates and reproduction in the spring flowering species Trillium erectum (Liliaceae)

We investigated the impact of overstory tree leaf phenology on growth rates, carbon allocation pattern, and fruit characteristics in the spring flowering species, Trillium erectum (Liliaceae). Air temperature, overstory canopy closure, and T. erectum phenology were monitored at three locations following a latitudinal gradient in Québec, Canada. Northern sugar maple trees leaf out at cooler temperatures than more southern populations, while Trillium development was initiated at the same soil temperature irrespective of the latitude. Therefore, in northern areas, the time between initiation of T. erectum leaf expansion and canopy closure was shorter than in southern areas, which left less time for northern plants to accumulate reserves before canopy closure. Differences in growth patterns were noted between T. erectum populations. From a south-north gradient, investment to reproduction, total plant biomass, and annual growth rate decreased, while specific leaf area and stem height increased, indicating shade acclimation. The length of the high light period in early spring seems to be a determinant for spring flowering plants' growth and reproduction and may explain the northern distribution limit of some of these species.     

Mechanisms of reproductive isolation of interspecific hybridization between Trillium camschatcense and T. tschonoskii (Melanthiaceae)

Reproductive isolation plays a significant role in the prevention of gene flow between different plant species. Isolation factors can vary, acting either pre‐ or postzygotically. Trillium camschatcense and T. tschonoskii are herbaceous perennials which frequently grow together in Hokkaido, Japan. Natural hybrid formation, T. × hagae, between these species is common, and occurs asymmetrically with T. camschatcense as the maternal parent and T. tschonoskii as the paternal. Here, we examined the efficiency of each reproductive isolation factor to clarify which factor was responsible for the frequency and asymmetry of the hybridization. We found that prezygotic barriers, self fertilization and conspecific pollen precedence, are major isolation factors in both parental species, and that T. tschonoskii as a maternal parent has more effective prezygotic barriers than T. camschatcense. In addition, hybrids with T. tschonoskii as the maternal parent were not observed to reach the flowering stage. We concluded that prezygotic isolation factors in the both species act as main barriers to prevent natural hybridization, and that asymmetry of the isolating barriers between these species would promote T. camschatcense as the maternal parent of the hybrids.     

Selfing inNarthecium ossifragum (Melanthiaceae)

Hagerup's (1950) observations of rain pollination inNarthecium ossifragum are commonly admitted. Nevertheless,Daumann (1970) stated that pollen and stigmas suffer considerable damage from rain impact. Experimental pollinations and pollen observations were conducted at a single site in the Upper Ardennes, Belgium, during three years. Rain water had a deleterious effect on both fruit and seed set and on pollen germination. Fruit and seed set differed among years mainly due to rainy weather. Fruit set also varied among experimental treatments. Pollination trials showed thatNarthecium ossifragum is self-compatible and autofertile. Emasculation had a deleterious effect and caging had no effect on fertility.     

The effect of pollinators,predators, and energy constraints on the floral ecology and evolution of Trillium erectum

Summary The reproductive success and energy budgets of single and multiflowered plants of Trillium erectum L. (Liliaceae) were assessed over a period of 4 years. Plants with more than one flower were found to attract more pollinators, to be less vulnerble to predation by Tortricid moths, and to produce more seeds than single flowered plants. Despite the reproductive superiority exhibited by multiflowered plants, most plants (85%) in the population studied were single flowered. It is shown that even though the insect interactions make it advantageous to produce more than one flower, most plants lack the energy to do so. The optimality approach which has guided previous studies of this kind is criticzied.     

Differentiation Patterns of Reproductive Systems in the Genus Trillium

Abstract The differentiation patterns of reproductive systems, including breeding and pollination systems, and perennation strategies of the genus Trillium were critically studied. The pedicellate- and sessile-flowered species with different floral morphologies and ecological distributions exhibited distinct modes of reproductive systems in relation to their habitat conditions. The pedicellate-flowered species occurring in stable mesic temperate deciduous forests reproduce exclusively by seeds, while sessile-flowered species growing in the alluvial flood plain habitats depend to a large extent on vegetative reproduction to compensate for the insufficient sexual reproduction under ecologically unstable conditions, e.g., frequent disturbance due to flooding. These sessile-flowered species are characterized by low ovule numbers, subsequent low seed outputs and low fecundity levels. In contrast to the sessile-flowered species, the pedicellate-erect-flowered species showed very high seed setting rates of over 50%. Breeding experiments for four Japanese species suggest that, although they substantially possess the capability of both inbreeding and outbreeding, the high fecundity levels are maintained by predominant inbreeding system. Furthermore, resource levels in the stock organs of these species obviously determine fecundity levels. As a consequence the amount of reserved food in the rhizomes transferred to the next season is determined, which gurantees a continuous reproductive activity of a given individual plant.     

Self-Incompatibility and Male Fertilization Success in Phillyrea angustifolia (Oleaceae)

Androdioecy is a rare breeding system in which low male frequency is expected in populations because males require a strong increase in their fertility to be maintained by selection. Phillyrea angustifolia L. has previously been reported as possibly functionally androdioecious. However, 1&rcolon;1 sex ratios have been reported and suggest functional dioecy. In this article, we compared both pollen tube growth and siring success of male and hermaphrodite pollen in two single-donor pollination experiments. We verified at both pre- and postzygotic levels that hermaphrodites produce functional pollen. Self-incompatibility was also clearly established. However, pollen from hermaphrodites was less efficient than male pollen. The probability of a pollen tube growing through the style was higher for male than for hermaphrodite pollen donors, and males sired twice as many fruits as hermaphrodites. The twofold male advantage in relative fecundity was mainly because of lower pollen fertility of hermaphrodites and possible cross-incompatibility among hermaphrodites.     

The evolution of self-compatible and self-incompatible populations in a hermaphroditic perennial, <Emphasis Type="Italic">Trillium camschatcense</Emphasis> (Melanthiaceae)

The evolution of selfing from outcrossing ancestors is known to have occurred repeatedly in angiosperms. Theoretical studies have argued that the transition from outcrossing to selfing is accomplished more easily than the reverse case, and phylogenetic analyses involving self-compatible (SC) and self-incompatible (SI) species has basically supported this assumption. The evolutionary direction of self-compatibility and self-incompatibility was examined in Trillium camschatcense, which contains geographically widespread SC populations, and restricted SI populations. Ecological surveys have revealed that the SC populations were suitable for outcrossing, and selfing in these populations did not confer any fitness advantage. Since reproductive fitness indicates the possibility of an evolutionary shift from self-compatibility to self-incompatibility, the phylogenetic relationships of SI and SC populations of T. camschatcense were investigated based on cpDNA variations and nuclear DNA microsatellite polymorphisms. Although phylogenetic analyses did not provide credible evidence to determine evolutionary direction, the SI populations turned out to be monophyletic with extremely low genetic differentiation. Based on these results, we proposed two possible scenarios for the evolutionary backgrounds of SI and SC populations in T. camschatcense. The plausibility of each scenario was evaluated based on the reproductive and geographical features of the mating systems. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sexual Attraction and Mating Patterns in Cylindrocorpus longistoma and C. curzii (Nematoda: Cylindrocorporidae)

Females of Cylindrocorpus longistoma and C. curzii excrete attractants which probably function to bring the sexes together before mating. Intraspecific and interspecific heterosexual and homosexual pairing experiments showed the attractants to be species specific as well as sex specific. Observations on mating behavior support the hypothesis that sexual attraction and copulation require independent stimuli.     

Seed dispersal in Erythronium grandiflorum (Liliaceae)

Primary and secondary seed dispersal was investigated for the glacier lily Erythronium grandiflorum in the Colorado Rocky Mountains. These heavy seeds have no obvious adaptations for biotic or abiotic dispersal, but can be thrown short distances when the dehiscent fruits are shaken by wind. We used sticky traps to measure primary transport of seeds up to 1 m away from individual plants. A seed cafeteria experiment examined the role of ants and rodents in secondary seed transport. Primary dispersal by wind was positively skewed and median transport distances were influenced by variation in plant height. Secondary dispersal was negligible compared to Viola nuttallii, an elaiosome-bearing species. Thus, seed dispersal was highly restricted in E. grandiflorum, and a 1 m radius encompassed the modal section of the seed dispersal curve. The seed dispersal component of gene flow was quantified and combined with previous measurements of pollen flow to yield a more complete estimate of Wright's neighborhood size, N _e, for E. grandiflorum. The lack of a special seed dispersal mechanism in E. grandiflorum is discussed in terms of a source-sink model for seedling establishment with respect to distance from the parental plants.     

Patterns of Inheritance of Mating Signals in Sailfin and Shortfin Mollies (Poeciliidae: Poecilia: Mollienesia)

Divergence in mating signals is a primary factor leading to reproductive isolation, and thus, speciation. However, the genetic changes underlying such divergence are poorly understood, especially in vertebrates. We used two species of poeciliid fishes, Poecilia velifera and P. mexicana , to explore the link between genes and mating behaviors that has resulted in pre-mating reproductive isolation between these species. Using backcross hybrids created from the F1 male offspring of reciprocal interspecific crosses between a sailfin molly ( P. velifera ) and a shortfin molly ( P. mexicana ), we examined the effects of Y-linkage and autosomal contributions on the expression of two male mating behaviors: courtship displays and gonopodial thrusts. The F1 hybrid males displayed a strong influence of sire on courtship display rates, with F1 males sired by the sailfin species showing courtship display rates that were up to three times higher than the rates of displays performed by F1 males sired by the shortfin species. These results suggest a Y-linked genetic effect on the expression of courtship display behavior. Comparisons between backcross hybrid males with sailfin Y-chromosomes or shortfin Y-chromosomes suggested that the interaction of autosomal genes also influences the inheritance of courtship display rates. Sailfin autosomal genes significantly increased the probability of performing courtship displays for hybrid males, and increased display rate for males from the sailfin Y-chromosome line. Autosomal genes had less of an impact on gonopodial thrusting behavior, however, thrust rates did significantly decrease with an increasing proportion of sailfin autosomes in males from the shortfin Y-chromosome line. These results suggest that the inheritance of species differences in mating signals between shortfin and sailfin mollies involves both genes found on the Y-chromosome and autosomal gene influences on their expression.     

Transformation of lisianthus (Eustoma grandiflorum)

Transformed plants from three cultivars of Eustoma grandiflorum (lisianthus) were produced by cocultivating young leaf pieces with Agrobacterium tumefaciens strain A722 containing the binary vectors pKIWI110 and pLN26. Both vectors contain the selectable marker gene for neomycin phosphotransferase II. pKIWI110 also contains the reporter gene for β-d-glucuronidase, and pLN26, the chalcone synthase antisense gene. Southern DNA analysis revealed that all the kanamycin-resistant transformants tested contained copies of the transgenes integrated in their genome. The two plants transformed with pKIWI110 show β-d-glucuronidase expression in their mature leaves and selected transformants passed on the kanamycin-resistant phenotype to the F1 generation. Received: 8 January 1997 / Revision received: 12 May 1997 / Accepted: 3 June 1997