Breeding Systems in American and Asian Trillium Species by Means of Chromosome Analyses

Abstract Species of Trillium have a disjunct distribution occurring in both North America and eastern Asia. In North America all 36 species are diploid. The 11 species of eastern Asia, however, include only a single diploid with all the other species being polyploids. Why do different patterns of speciation develop in North America and in eastern Asia? The breeding systems of populations in the North American T. erectum, T. grandiflorum and T. ovatum , and in Asian T. kamtschaticum were investigated by estimating the inbreeding coefficient from cold-induced banding patterns which reveal homozygotes and heterozygotes. From the analyses of the inbreeding coefficients, T. erectum, T. grandiflorum and the Pacific coastal species, T. ovatum are predominantly inbreeding species. T. ovatum populations from the Rocky Mountain region are outbreeders. However the Japanese species, T. kamtschaticum has a mixture of outbreeding and inbreeding among populations. The development of polyploid systems in Asia is possibly the result of the diversity of the breeding systems among the populations. The shift from outbreeding to inbreeding appears to be an important key step in the occurrence of poliploids by hybridization between the different species.     

Structure and Dynamics of Natural Populations of Polyploid Trilliums. V. Predominant Self-pollination in Trillium apetalon-T. smallii Association

Abstract Predominant self-pollination was confirmed in mixed populations of Trillium apetalon (4x = 20, SSUU) and T. smallii (6x = 30, K₁K₁SSUU). In pollination tests conducted on both species from the Muroran (Mr) population, many good seeds were obtained from bagged flowers as well as open-pollinated intact flowers. In striking contrast, fruits from castrated open-pollinated flowers failed to give any good seeds. In T. smallii in the Usu (Us) population, the same results were obtained for bagged and open-pollinated intact flowers. On the other hand, fruits from castrated open-pollinated flowers yielded only a few good seeds. Although both species were experimentally cross-compatible in both directions, no interspecific hybrids have so far been found in nature. It may be possible that both species are isolated by predominant self-pollinatioin even though their flowering seasons coincide.     

Pollinators exert selection on floral traits in a pollen-limited,narrowly endemic spring ephemeral

Premise

Floral traits are frequently under pollinator-mediated selection, especially in taxa subject to strong pollen-limitation, such as those reliant on pollinators. However, antagonists can be agents of selection on floral traits as well. The causes of selection acting on spring ephemerals are understudied though these species can experience particularly strong pollen-limitation. I examined pollinator- and antagonist-mediated selection in a narrowly endemic spring ephemeral, Trillium discolor.

Methods

I measured pollen limitation in T. discolor across two years and evaluated its breeding system. I compared selection on floral traits (display height, petal size, petal color, flowering time) between open-pollinated, and pollen-supplemented plants to measure the strength and mode of pollinator-mediated selection. I assessed whether natural levels of antagonism impacted selection on floral traits.

Results

Trillium discolor was self-incompatible and experienced pollen limitation in both years of the study. Pollinators exerted negative disruptive selection on display height and petals size. In one year, pollinator-mediated selection favored lighter petals but in the second year pollinators favored darker petals. Antagonist damage did not alter selection on floral traits.

Conclusions

Results demonstrate that pollinators mediate the strength and mode of selection on floral traits in T. discolor. Interannual variation in the strength, mode, and direction of pollinator-mediated selection on floral traits could be important for maintaining of floral diversity in this system. Observed levels of antagonism were weak agents of selection on floral traits.     

Pollen-mediated gene dispersal within continuous and fragmented populations of a forest understorey species, Trillium cuneatum

Pollen movement plays a critical role in the distribution of genetic variation within and among plant populations. Direct measures of pollen movement in the large, continuous populations that characterize many herbaceous plant species are often technically difficult and biologically unreliable. Here, we studied contemporary pollen movement in four large populations of Trillium cuneatum. Three populations, located in the Georgia Piedmont, are exposed to strong anthropogenic disturbances, while the fourth population, located in the Southern Appalachian Mountains, is relatively undisturbed. Using the recently developed TwoGener analysis, we extracted estimates of the effective number of pollen donors (N(ep)), effective mating neighbourhood size (A(ep)) and the average distance of pollen movement (delta) for each population. We extended the TwoGener method by developing inference on the paternal gametic contribution to the embryo in situations where offspring genotypes are inferred from seeds and elaiosomes of species with bisporic megagametogenesis. Our estimates indicate that maternal plants do not sample pollen randomly from a global pool; rather, pollen movement in all four populations is highly restricted. Although the effective number of pollen donors per maternal plant is low (1.22-1.66) and pollen movement is highly localized in all populations, N(ep) in the disturbed Piedmont populations is higher and there is more pollen movement than in the mountains. The distance pollen moves is greater in disturbed sites and fragmented populations, possibly due to edge effects in Trillium habitats.     

Stigmatic Self-Incompatibility and Mating Patterns in Trillium grandiflorum and Trillium erectum(Melanthiaceae)

Post-pollination processes governing mating patterns in Trillium,a well-known genus of insect-pollinated woodland herbs, arepoorly understood. Mechanisms influencing outcrossing were investigatedin T. grandiflorum and T. erectum, two widespread species nativeto eastern North America. In southern Ontario, Canada, the twospecies are often sympatric; they flower in early May, and arepollinated by different assemblages of insects. Controlled cross-and self-pollinations and structural observations of pollengermination and pollen tube growth were conducted to determinewhether the two species possess a self-incompatibility (SI)system and, if so, the specific site(s) of self-rejection. Controlledpollinations indicated that both species set significantly moreseeds from cross-pollination than self-pollination, implicatingthe action of SI. This was confirmed by structural studies whichdemonstrated that self-recognition and rejection reactions occurredon dry-type stigmatic papillae. Observations of pollen hydrationrevealed that self-rejection was rapid, being initiated within10 min of pollination and prior to pollen tube emergence. Finalself-rejection resulted in failure of pollen tube growth atthe base of stigmatic papillae. SI was expressed more weaklyin T. erectum and thereby resulted in considerable self-seedset in some individuals . Estimates of outcrossing rates usingallozyme markers indicated that T. erectum displayed a mixed-matingsystem whereas T. grandiflorum was more highly outcrossed. Structuralstudies of pollen traits indicated that the two species differedwith respect to the size of grains and their aggregation withimplications for pollen dispersal and mating. The ecologicaland evolutionary implications of the variable expression ofSI in Trillium are discussed. Copyright 2001 Annals of BotanyCompany Trillium grandiflorum, Trillium erectum, self-incompatibility, mating     

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.     

Floral density,pollen limitation,and reproductive success in<Emphasis Type="Italic"> Trillium grandiflorum</Emphasis>

Decreases in floral density can disrupt mutualistic interactions between plants and their pollinators, and decrease reproductive success. I addressed the relationship between floral density and plant reproductive success using two experimental approaches: a pollen supplementation experiment in 12 populations of Trillium grandiflorum that naturally varied in floral density, and a transplant experiment in which floral density was manipulated in plots at four experimental sites. In the pollen supplementation experiments, the degree of pollen limitation, in terms of fruit set and seed set, decreased with floral density. Further, in the experimental sites, plant reproductive success increased asymptotically with floral density. These results demonstrate the value of simultaneously conducting experiments in both experimental sites and natural populations to understand how population density influences plant reproductive success. Factors that reduce the density of this perennial herb, such as habitat fragmentation and herbivory by white-tailed deer (Odocoileus virginianus), should be expected to limit its reproduction.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

A field study of seed germination in the endangered Trillium reliquum Freeman (Trilliaceae)

Previous studies examining the seeds of most Trillium species have reported double dormancy, a type of seed dormancy where two cold periods and one warm period are needed for complete germination. In the present paper, we describe a field study examining the federally endangered Trillium reliquum Freeman (Trilliaceae) in which moderate to high numbers of seeds germinated after one winter following seed production. Sixteen baskets with seeds were placed in four T. reliquum populations (four baskets in each population) in Georgia, USA, in June 2005. In spring 2006, all seed baskets contained seedlings. Germination percentages ranged from 33.3 to 83.3% across sites with a mean of 56.9 ± 3.9%. Trillium reliquum had higher germination percentages compared with other field‐based germination studies with other Trillium species. Our findings will inform future demographic studies of T. reliquum and suggest that double dormancy in seeds may not be as widespread as previously reported within the genus Trillium.     

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.     

Life History Studies on the Genus Trillium (Liliaceae) V. Reproductive Biology and Survivorship of Three Declinate-flowered Species

Absract Three pedicellate-declinate-flowered species of Trillium (Liliaceae), T. vaseyi and T. flexipes , were studied for their life history characteristics, e.g., stage class structures of natural populations and reproductive features, including energy allocation to reproductive activities. The populations structures of all three species showed similar depletion structures characterized by a conspicuous decrease of individuals in the small juvenile stages, as was also observed in pedicellate-erect-flowered Trillium species However, with respect to reproductive characteristics, these three declinate-flowered species showed different features from erect-flowered species, although they belong to the same pedicellate-flowered group. That is, these declinate-flowered Trillium species exhibited low seed setting rates of 30% in T. catesbaei , 45% in T. vaseyi and 34% in T. flexipes , suggesting that they possess different mating systems from erect-flowered species which showed high seed setting rates of 50–90%.     

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.     

Pollinator‐mediated phenotypic selection does not always modulate flower size and number in the large‐flowered Mediterranean shrub Cistus ladanifer (Cistaceae)

Flower size and number usually evolve under pollinator‐mediated selection. However, hot, dry environments can also modulate display, counteracting pollinator attraction. Increased pollen deposition on larger flower displays may not involve higher female fitness. Consequently, stressful conditions may constrain flower size, favouring smaller‐sized flowers. The large‐flowered, self‐incompatible Mediterranean shrub Cistus ladanifer was used to test that: (1) this species suffers pollen limitation; (2) pollinators are spatially–temporally variable and differentially visit plants with more/larger flowers; (3) increased visits enhance reproduction under pollen limitation; (4) stressful conditions reduce female fitness of larger displays; and (5) phenotypic selection on floral display is not just pollinator‐mediated. We evaluated pollen limitation, related floral display to pollinator visits and fruit and seed production and estimated phenotypic selection. Flower size was 7.2–10.5 cm and varied spatially–temporally. Visitation rates (total visits/50 min) ranged from 0.26 to 0.43 and increased with flower size. Fruit set averaged 80% and seed number averaged 855, but only fruit set varied between populations and years. Selection towards larger flowers was detected under conditions of pollen limitation. Otherwise, we detected stabilizing selection on flower size and negative selection on flower number. Our results suggest that selection on floral display is not only pollinator‐dependent through female fitness in C. ladanifer. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 540–555.     

Genetic Structure in a Heterocyanic Population of Trillium sessile (Liliaceae)

Abstract Species of Trillium in the subgenus Phyllantherum are either polymorphic for flower color, or monomorphic for flower color and related to a polymorphic species. This leads to the suggestion that polymorphic species may be the progenitors for monomorphic ones. For this to be true, it must be demonstrated that genetic divergence among flower morphs can occur within polymorphic populations. Genetic structure was assessed in a population of T. sessile that contains a polymorphism for flower color. A survey of 11 enzyme systems using starch gel electrophoresis revealed three polymorphic loci: 6PGD-1, AAT-1 and AAT-2. Analysis of large and small scale spatial structure, stage classes, and flower color classes revealed significant genetic divergence in all instances. Spatial structure in the population is likely a result of genetic neighborhoods which can maintain populational variation via random genetic drift. Genetic divergence of the yellow flower color morph was probably initiated through genetic drift since the morph occurs in low frequencies. The results imply that the initial genetic divergence of species in the subgenus can arise within polymorphic populations.     

Pollen limitation and natural selection on floral characters in the yellow monkeyflower, Mimulus guttatus

In flowering plants, pollen limitation has been proposed to intensify selection on floral characters important in pollinator attraction, but may also select for traits that increase seed set through autonomous selfing. Here, a factorial design (+/- pollen addition, +/- pollinator removal) was used to investigate how the pollination environment affects selection on floral morphology via female fitness in a mixed-mating population of the yellow monkeyflower, Mimulus guttatus (Phrymaceae). Female fitness was strongly pollen-limited, with supplementally pollinated plants setting 37% more seeds than open-pollinated individuals. Strong positive selection was found on flower length, weak positive selection on flower width : length ratio and no selection on stigma-anther distance in both open-pollinated and supplementally pollinated treatments. By contrast, flowers with relatively narrow corollas and low stigma-anther distances were favored in the pollinator exclusion treatment. These results provide mixed support for the idea that pollen limitation intensifies selection on floral characters. Despite strong phenotypic selection, natural pollen limitation did not mediate selection on characters associated with either pollinator attraction or self-fertilization. However, the novel pattern of selection on severely pollen-limited plants suggests that reproductive assurance against pollinator loss may have been directly involved in the floral evolution of closely related selfing taxa.     

Pollination and reproduction of a self-incompatible forest herb in hedgerow corridors and forest patches

Habitat-corridors are assumed to counteract the negative impacts of habitat loss and fragmentation, but their efficiency in doing so depends on the maintenance of ecological processes in corridor conditions. For plants dispersing in linear habitats, one of these critical processes is the maintenance of adequate pollen transfer to insure seed production within the corridor. This study focuses on a common, self-incompatible forest herb, Trillium grandiflorum, to assess plant–pollinator interactions and the influence of spatial processes on plant reproduction in hedgerow corridors compared to forests. First, using pollen supplementation experiments over 2 years, we quantified the extent of pollen limitation in both habitats, testing the prediction of greater limitation in small hedgerow populations than in forests. While pollen limitation of fruit and seed set was common, its magnitude did not differ between habitats. Variations among sites, however, suggested an influence of landscape context on pollination services. Second, we examined the effect of isolation on plant reproduction by monitoring fruit and seed production, as well as pollinator activity and assemblage, in small flower arrays transplanted in hedgerows at increasing distances from forest and from each other. We detected no difference in the proportion of flowers setting fruit or in pollinator activity with isolation, but we observed some differences in pollinator assemblages. Seed set, on the other hand, declined significantly with increasing isolation in the second year of the study, but not in the first year, suggesting altered pollen transfer with distance. Overall, plants in hedgerow corridors and forests benefited from similar pollination services. In this system, plant–pollinator interactions and reproduction seem to be influenced more by variations in resource distribution over years and landscapes than by local habitat conditions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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.     

Effects of population size on reproductive success of the endangered and endemic species Primula merrilliana

The reproductive ability related to the population size of the endangered and endemic species Primula merrilliana Schltr.was investigated.In 26 populations observed,only four contain more than 500 flowering individuals,whereas most of them(53.8%) consist of less than 100 flowering individuals.Though the ratio of pin and thrum plants keeps its balance at 1:1 for all populations,the frequency of pin and thrum flowers was significantly biased in most small populations.Population size strongly affected reproductive success; plants in small populations produced significantly fewer flowers and fruits per plant and fewer seeds per fruit,and therefore fewer seeds per plant.The floral density was another important factor that influenced the reproductive success of P.merrilliana,because four main reproductive success parameters (fruits per plant,seeds per fruit,seeds per plant,and the proportion of flowers setting fruit) were all positively correlated with floral density.The size of plants and the number of leaves per plant (measure of habitat quality) were not influenced by the variation of population size,suggesting that the reduced fecundity in small populations may not be a consequence of lower habitat quality,inbreeding depression and pollen limitation as a result of less attractiveness in small populations are therefore likely explanations for these patterns.     

Variation of pollen limitation in the early flowering Mediterranean geophyte Narcissus assoanus (Amaryllidaceae)

A recent literature review indicates that pollen limitation of female fertility is a common feature of flowering plants. Despite the ecological and evolutionary significance of pollen limitation, most studies have only examined fertility in a single population at one time. Here we investigate pollen limitation of fruit and seed set in five populations of Narcissus assoanus, a self- sterile, insect-pollinated geophyte, over 2–3 years in southern France. In common with many early spring flowering plants, pollinator visitation to N. assoanus is often infrequent. Supplemental hand-pollination of flowers with outcross pollen significantly increased overall fruit and seed set by 11% and 19%, respectively. Four of the five populations experienced some pollen limitation during the study. For a given year, there was significant variation in pollen limitation among populations. Two of the populations were pollen limited in one year but not in other years in which they were studied. Seed:ovule ratios for open- and hand-pollinated flowers averaged 0.29 and 0.33, respectively. While hand pollination significantly increased the seed:ovule ratio, the low value obtained indicates that the majority of ovules in flowers do not mature seeds despite hand pollination. The role of genetic and environmental factors governing low seed:ovule ratios in N. assoanus is discussed. Received: 28 December 1999 / Accepted: 6 April 2000     

Floral morphology and reproductive success in the orchid Epipactis helleborine: regional and local across-habitat variation

 The terrestrial orchid Epipactis helleborine is a morphologically variable species with a wide distribution in Europe. It is pollinated by social wasps, and most populations show the morphological characteristics of outcrossing species. However, local predominantly selfing subspecies and varieties have been documented from drier habitats. To document geographic variation in floral morphology, ability to produce seeds through autogamy, and reproductive success in E. helleborine, we sampled 13 populations from three geographic regions along a latitudinal gradient of c. 1000 km from northern to southern Sweden. In the southernmost region, populations in dry and mesic habitats were compared. Supplemental hand-pollination was conducted to determine whether among-population variation in fruit set could be explained by differences in the natural level of pollination, and whether any relationship between floral morphology and fruit production could be explained by interactions with pollinators. Bagging experiments showed no evidence of autogamy in any of the study populations. Number of flowers, pollinia removal and fruit set varied significantly among populations but did not differ among regions. Pollinia removal was positively correlated with population size and both pollinia removal and fruit set were lower in dry than in mesic habitats. At the level of the individual plant, the number of pollinia removed increased more rapidly with flower number than did number of fruits produced. The hand-pollination experiment indicated that the positive relationship between number of flowers and fruit production was due to a higher degree of pollen limitation in plants with few flowers than in plants with many flowers. The experiment also showed that variation in the level of pollen limitation could only partly explain variation in fruit set among populations. Received November 6, 2001; accepted April 27, 2002 Published online: December 3, 2002