Increasing the germination percentage of a declining native orchid (Himantoglossum adriaticum) by pollen transfer and outbreeding between populations

• The declining native orchid Himantoglossum adriaticum H. Baumann is a European endemic of priority interest (92/43/ EEC, Annex II). Northern Italian populations of H. adriaticum are small and isolated, with depressed seed set. Given the important implications for plant population conservation, we tested the hypothesis that artificial pollen transfer (hand‐pollination) and outbreeding between populations increases fruit set and seed germination percentage.
• The background fruit set and in vitro germination rates were determined for ten reference populations. An artificial cross‐pollination experiment included (a) pollen transfer from one large population to two small and isolated populations; (b) pollen transfer between two small but not isolated populations; (c) within‐population pollen transfer (control). All seeds were sown on a modified Malmgren's medium and cultured in a controlled environment. Germination percentage was compared using a Kruskal‐Wallis anova .
• The background fruit set (mean = 18%) and germination (<5%) rates were consistently low across populations. Fruit set after hand‐pollination was consistently 100%. Pollen transfer from the largest population to smaller populations resulted in an increase in total germination ranging from 0.9% to 2.9%. The largest increase in germination occurred between small‐sized and less isolated populations (from 1.7% to 5.1%).
• The results of pollen transfer between the small populations are particularly encouraging, as the mean increase in germination was almost four times that of the control. Outbreeding can be considered a valuable tool to increase genetic flow and germination in natural populations, limit the accumulation of detrimental effects on fitness driven by repeated breeding with closely‐related individuals, thereby increasing the possibility of conservation of rare or endangered species.

     

Rainfall seasonality predicts the germination behavior of a tropical dry‐forest vine

Seed dormancy is considered to be an adaptive strategy in seasonal and/or unpredictable environments because it prevents germination during climatically favorable periods that are too short for seedling establishment. Tropical dry forests are seasonal environments where seed dormancy may play an important role in plant resilience and resistance to changing precipitation patterns. We studied the germination behavior of seeds from six populations of the Neotropical vine Dalechampia scandens (Euphorbiaceae) originating from environments of contrasting rainfall seasonality. Seeds produced by second greenhouse‐generation plants were measured and exposed to a favorable wet environment at different time intervals after capsule dehiscence and seed dispersal. We recorded the success and the timing of germination. All populations produced at least some dormant seeds, but seeds of populations originating from more seasonal environments required longer periods of after‐ripening before germinating. Within populations, larger seeds tended to require longer after‐ripening periods than did smaller seeds. These results indicate among‐population genetic differences in germination behavior and suggest that these populations are adapted to local environmental conditions. They also suggest that seed size may influence germination timing within populations. Ongoing changes in seasonality patterns in tropical dry forests may impose strong selection on these traits.     

Pollen source affects female reproductive success and early offspring traits in the rare endemic plant Polemonium vanbruntiae (Polemoniaceae)

Understanding the relative magnitudes of inbreeding and outbreeding depression in rare plant populations is increasingly important for effective management strategies. There may be positive and negative effects of crossing individuals in fragmented populations. Conservation strategies may include introducing new genetic material into rare plant populations, which may be beneficial or detrimental based on whether hybrid offspring are of increased or decreased quality. Thus, it is important to determine the effects of pollen source on offspring fitness in rare plants. We established pollen crosses (i.e. geitonogamous‐self, autonomous‐self, intrasite‐outcross, intersite‐outcross and open‐pollinated controls) to determine the effects of pollen source on fitness (seeds/fruit and seed mass) and early offspring traits (probability of germination, number of leaves, leaf area and seedling height) in the rare plant Polemonium vanbruntiae. Open‐pollinated, intrasite‐outcross and geitonogamous‐self treatments did not differ in fitness. However, plants receiving autonomous‐self pollen had the lowest fitness and the lowest probability of seed germination. Intersite‐outcross plants contained fewer seeds/fruit, but seeds germinated at higher frequencies and seedlings were more vigorous. We also detected heterosis at the seed germination stage. These data may imply that natural populations of P. vanbruntiae exhibit low genetic variation and little gene flow. Evidence suggests that deleterious alleles were not responsible for reduced germination; rather environmental factors, dichogamy, herkogamy and/or lack of competition among pollen grains may have caused low germinability in selfed offspring. Although self‐pollination may provide some reproductive assurance in P. vanbruntiae, the result is a reduction in germination and size‐related early traits for selfed offspring.     

Effects of population density on male and female reproductive success in the wind-pollinated, wind-dispersed tree species Betula maximowicziana

The effects of population density on male and female reproductive success of Betula maximowicziana were evaluated in two mixed and two post-fire stands, with various population densities, ranging from 1.9 to 300.0 trees per ha, in central Hokkaido, Japan. First, we investigated ecological determinants of reproductive success (seed set and germination) of both seeds collected from the trees (tree seeds) and dispersed seeds collected from seed traps (dispersed seeds). We then evaluated the effects of population density on seed set and germination of tree seeds and dispersed seeds using a generalized linear mixed model (GLMM). Subsequently, we genotyped 950 seeds collected from mother trees and 940 seeds trapped after dispersal derived from tree seeds and dispersed seeds, respectively, using eleven microsatellite loci. Using the acquired data, we then evaluated the outcrossing rate and effective number of pollen donors (N _ep) of the tree seeds, and the genetic structure of both pollen pools and dispersed seed populations. The seed set and germination rate of dispersed seeds was significantly lower both in the lowest-density stand and in the highest-density stand. The GLMM revealed that seed set and germination rates of dispersed seeds may be maximal at approximately 120 trees per hectare (optimal density). Outcrossing rates were consistently high (t _m = 0.995), regardless of the population density. In contrast, N _ep was lower in the lowest-density stands. Significant genetic structure of the dispersed seed population was found in two low-density stands, probably due to the limitation of overlapping seed shadows.     

Adaptation to habitat in Aquilegia species endemic to Sardinia (Italy): Seed dispersal,germination and persistence in the soil

Abstract

The autecology of the Sardinian endemics Aquilegia barbaricina Arrigoni et Nardi and A. nugorensis Arrigoni et Nardi were investigated. Peaks of anthesis and seed dispersal were recorded for five populations occurring in two distinct habitats, one riparian and one rupicolous. Germination tests were carried out on seed lots belonging to each population by sowing seeds at 10, 15, 20, 25 and 25/15°C. In addition, seeds were incubated for 2 months at either 25°C (summer), 5°C (winter) or 25°C for 2 months plus 2 months at 5°C (summer followed by winter–SW), and then moved to the germination temperatures. Embryo measurements were taken during pre-treatments and germination. Experimental seed burials were carried out for two populations of each species. Both species dispersed in summer. The population of A. nugorensis occurring on rocky outcrops differed in phenology from both the other A. nugorensis population from riparian vegetation and from A. barbaricina. Both species showed morphophysiological seed dormancy, with <50% germination under laboratory conditions. All riparian populations germinated only after the SW pre-treatment, while the rupicolous population germinated at 25°C, without any pre-treatment. Low germination percentages were observed in the experimental seed burials, suggesting the ability for both species to form a persistent soil seed bank.     

Pollen limitation and Allee effect related to population size and sex ratio in the endangered Ottelia acuminata (Hydrocharitaceae): implications for conservation and reintroduction

Small populations may suffer more severe pollen limitation and result in Allee effects. Sex ratio may also affect pollination and reproduction success in dioecious species, which is always overlooked when performing conservation and reintroduction tasks. In this study, we investigated whether and how population size and sex ratio affected pollen limitation and reproduction in the endangered Ottelia acuminata, a dioecious submerged species. We established experimental plots with increasing population size and male sex ratio. We observed insect visitation, estimated pollen limitation by hand‐pollinations and counted fruit set and seed production per fruit. Fruit set and seed production decreased significantly in small populations due to pollinator scarcity and thus suffered more severe pollen limitation. Although frequently visited, female‐biased larger populations also suffered severe pollen limitation due to few effective visits and insufficient pollen availability. Rising male ratio enhanced pollination service and hence reproduction. Unexpectedly, pollinator preferences did not cause reduced reproduction in male‐biased populations because of high pollen availability. However, reproductive outputs showed more variability in severe male‐biased populations. Our results revealed two component Allee effects in fruit set and seed production, mediated by pollen limitation in O. acuminata. Moreover, reproduction decreased significantly in larger female‐biased populations, increasing the risk of an Allee effect.     

Marked differences in seed dormancy in two populations of the Mediterranean shrub,Cistus albidus L.

Background: Population structure and dynamics in natural ecosystems can be affected by seed viability and dormancy. However, how the endogenous contents of phytohormones and vitamin E in seeds relative to the environment affect viability and dormancy is not yet fully understood.

Aims: We studied seed viability and germination capacity in two populations of the Mediterranean shrub, Cistus albidus, exposed to contrasting environmental conditions.

Methods: We measured seed viability and germination capacity and endogenous contents of abscisic acid (ABA), gibberellins and vitamin E in seeds collected from two populations, in two environmentally contrasting environments in north-eastern Spain.

Results: Plants growing in the natural site produced seeds with lower germination percentage but similar viability, indicating higher seed dormancy. Enhanced seed dormancy was paralleled with higher contents of ABA and lower contents of gibberellins (GAs). Contents of tocopherols and tocotrienols were higher in seeds of the natural population. Tocopherol contents in seeds correlated positively with the ratio living:total aerial biomass.

Conclusions: Two Mediterranean populations of C. albidus growing in two sites with contrasting environmental conditions showed marked differences in seed dormancy and germination, which may be explained, at least in part, by differences in seed hormonal contents.     

Variation of pollination and resource limitation in a low seed-set tree, Liriodendron chinense (Magnoliaceae)

Pollen limitation and resource limitation have been documented as the major factors responsible for plants commonly producing more ovules than seeds, but few studies have examined pollen deposition directly in natural populations at different sites and times. We investigated the causes of low seed set in four populations of Liriodendron chinense (Magnoliaceae), an insect‐pollinated endangered tree endemic to southern China, over 2–3 years. One pistil potentially produces two ovules. The number of pistils per flower varies among populations, but in three of the four populations the variation in a given population was not significantly different among years. Overall, populations with higher pistil numbers tend to set more seeds per flower, but a positive correlation between pistil numbers and seed production per flower was observed in only one of the four populations. The numbers of pollen grains deposited per stigma varied from 0 to 60. The proportion of pollinated stigmas per flower ranged from 44% to 88% among populations and years. The numbers of pollen grains deposited per stigma and the percentages of pollinated stigmas were significantly different between populations, and two populations showed significant differences between years. A positive correlation between stigmatic pollen load and seed set was sought in ten population‐by‐year combinations but, in a given population, high stigmatic pollen loads did not always result in high seed set. Examination of pollen deposition, pistil and seed production over several sites and years showed that in addition to pollination, other factors such as resource or genetic loads were likely to limit the (lower than 10%) seed set in L. chinense. It appears that small, isolated populations experience severe pollination limitation; one population studied had seed/ovule ratios of 0.84% and 1.88% in 1995 and 1996. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 140 , 31–38.     

POLLINATOR EFFECTIVENESS AND SEED SET IN POPULATIONS OF AGALINIS STRICTIFOLIA (SCROPHULARIACEAE)

Examining variation in pollinator effectiveness and seed production resulting from single pollinator visits can provide a deeper understanding of how pollinators may influence reproduction in plant populations. When comparing populations, differences in the number of seeds produced from single pollinator visits to flowers may not always be attributable to differences in pollen deposition, but rather to differences in plant fecundity or resource availability. Pollinator effectiveness and seed production were studied for two populations over a 4-year period and were measured using single bee visit manipulations of flowers. No significant difference in pollinator effectiveness (pollen deposited on stigmas) was observed between the two populations. However, a significant difference between the two populations was observed in the number of seeds produced per flower. The Wellhouse population produced approximately three times as many seeds/flower from a single pollinator visit as did the Firefly Meadow population. Within each population, pollinators (Bombus pennsylvanicus and Apis mellifera) did not differ in the number of pollen grains deposited on stigmas or seeds produced per flower from single visit experiments. Differences in plant density, pollen viability, and ovules per flower also could not account for a significant amount of the variation. A resource augmentation experiment (water and fertilizer application) resulted in a decrease in seeds per flower per bee visit for the water treatment at the Wellhouse population only. For both populations, pollen deposition, pollen viability, and ovules per flower were unaffected by the resource augmentation. Alternative possibilities for the observed differences in seeds per flower per bee visit are discussed.     

Genetic rescue by distant trees mitigates qualitative pollen limitation imposed by fine‐scale spatial genetic structure

Restricted seed dispersal frequently leads to fine‐scale spatial genetic structure (i.e., FSGS) within plant populations. Depending on its spatial extent and the mobility of pollinators, this inflated kinship at the immediate neighbourhood can critically impoverish pollen quality. Despite the common occurrence of positive FSGS within plant populations, our knowledge regarding the role of long‐distance pollination preventing reproductive failure is still limited. Using microsatellite markers, we examined the existence of positive FSGS in two low‐density populations of the tree Pyrus bourgaeana. We also designed controlled crosses among trees differing in their kinship to investigate the effects of increased local kinship on plant reproduction. We used six pollination treatments and fully monitored fruit production, fruit and seed weight, proportion of mature seeds per fruit, and seed germination. Our results revealed positive FSGS in both study populations and lower fruit initiation in flowers pollinated with pollen from highly‐genetically related individuals within the neighbourhood, with this trend intensifying as the fruit development progressed. Besides, open‐pollinated flowers exhibited lower performance compared to those pollinated by distant pollen donors, suggesting intense qualitative pollen limitation in natural populations. We found positive fine‐scale spatial genetic structure is translated into impoverished pollen quality from nearby pollen donors which negatively impacts the reproductive success of trees in low‐density populations. Under this scenario of intrapopulation genetic rescue by distant pollen donors, the relevance of highly‐mobile pollinators for connecting spatially and genetically distant patches of trees may be crucial to safeguarding population recruitment.     

The functional ecology of gynodioecy in Eritrichum aretioides (Boraginaceae), the alpine forget-me-not

Eritrichum aretioides is a gynodioecious species with female and hermaphrodite individuals. In populations on Pennsylvania Mountain in central Colorado (USA), the frequency of females ranges from 22 to 41%. Flower number and the number of seeds produced per flower were similar in hermaphrodites and females. However, hermaphrodites produced larger flowers, while females produced larger seeds (P < 0.05 for both). In the field, seed germination was higher for seeds from females than for seeds from hermaphrodites (20 vs. 9% germination; P < 0.05). Unvisited flowers and open-pollinated flowers of hermaphrodites had similar pollen receipt (approx 20 pollen grains per stigma), but seed set following autogamous pollination was significantly lower than seed set following natural pollination. This finding indicates that hermaphrodites have a barrier to selfing and implies that the larger seed size and greater establishment advantage of offspring from females is unlikely to have resulted from female outcrossing advantage. Rather, differences in the quality of seed progeny between morphs probably reflect a trade-off in sexual allocation or pleiotropic effects of the sex-determining genes.     

INBREEDING DEPRESSION IN ANDRODIOECIOUS POPULATIONS OF DATISCA GLOMERATA (DATISCACEAE)

Several workers have suggested that the rarity of androdioecy (the presence of males and hermaphrodites in a breeding population) in nature is due to the large fitness gain required by male plants in order to be maintained by selection. As part of an ongoing investigation of this hypothesis, we tested the effects of selfing on fitness in functionally androdioecious populations of Datisca glomerata. We compared progeny from self-fertilizations, cross-fertilizations with pollen from male plants only, cross-fertilizations with pollen from hermaphrodite plants only, and open-pollinated flowers for several measures of progeny fitness including seed weight, germination rate, and seedling weight. Significant inbreeding depression was observed for androdioecious populations of D. glomerata for both seed and seedling weights. However, no significant differences were observed across treatments for seed germination percentages. The observation of significant levels of inbreeding depression in this study, combined with prior evidence of threefold greater pollen production by males, may at least partially account for the large fitness increase required by males to be maintained by selection.     

Natural levels of pollination intensity and effects of pollen loads on offspring quality in females of Thalictrum pubescens (Ranunculaceae)

Females of Thalictrum pubescens produce stamens containing sterile pollen. Earlier studies have shown the presence of stamens does not increase seed set through increased visitation by insects. Insects may, however, increase pollen deposition on stigmas and increase pollen competition. This paper examines: 1) pollen deposition levels in natural populations and 2) whether larger pollen loads lead to higher quality offspring. The majority of stigmas in two populations received less than 15 grains, but a small proportion had large loads. The latter may represent the occasional insect visit. These visits may provide the opportunity for pollen tube competition. In the greenhouse, flowers receiving heavy-pollen loads had higher seed set than flowers receiving light-pollen loads. Seeds from the two treatments were the same mass, had similar germination times, and seedlings had the same mass. In conclusion, it does not appear that pollen tube competition can account for the maintenance of stamens in females of T. pubescens.     

Pollen limitation in the endangered Chinese endemic species Sinocalycanthus chinensis

Pollen limitation negatively impacts endangered and endemic plants with small fragmented populations, such as Sinocalycanthus chinensis, an endangered plant endemic to China. In this study, we analyzed the pollen limitation of the S. chinensis Damingshan (DMS) population in 2006, 2009, and 2010, and crossed plants with mates separated by different distances, both within and between populations. The DMS population exhibited strong pollen limitation in fruit set, seed set, and seeds per fruit in 2006, 2009, and 2010. The average accumulated pollen limitation (for fruit set times seeds per fruit) was 0.510 ± 0.180. Progeny crossed with pollen from intermediate neighboring plants within the same population (separated by 30–50 m from pollen recipients) had the lowest fitness. No optimal outcrossing distance was found within the DMS population. Progeny from crosses with the Shunxiwu (SXW) and Daleishan (DLS) populations performed relatively better, while those from crosses with Qingliangfeng (QLF) and Longxushan (LXS) populations performed worse. Compared with average reproductive success, outbreeding depression was found in progeny from crosses with the LXS and QLF populations. Reproductive success from pure self‐pollination indicated S. chinensis is self‐compatible. Geitonogamous selfing increased reproductive success. Based on geitonogamous selfing, the proportion of selfed offspring was relatively high. These results provide basic references for the conservation of this species.     

Consequences of inbreeding for offspring fitness and gender in Silene vulgaris,a gynodioecious plant

Abstract In gynodioecious plants, hermaphrodite and female plants co‐occur in the same population. In these systems gender typically depends on whether a maternally inherited cytoplasmic male sterility factor (CMS) is counteracted by nuclear restorer alleles. These restorer alleles are often genetically dominant. Although plants of the female morph are obligatorily outcrossing, hermaphrodites may self. This selfing increases homozygosity and may thus have two effects: (1) it may decrease fitness (i.e. result in inbreeding depression) and (ii) it may increase homozygosity of the nuclear restorer alleles and therefore increase the production of females. This, in turn, enhances outcrossing in the following generation. In order to test the latter hypothesis, experimental crosses were conducted using individuals derived from four natural populations of Silene vulgaris, a gynodioecious plant. Treatments included self‐fertilization of hermaphrodites, outcrossing of hermaphrodites and females using pollen derived from the same source population as the pollen recipients, and outcrossing hermaphrodites and females using pollen derived from different source populations. Offspring were scored for seed germination, survivorship to flowering and gender. The products of self‐fertilization had reduced survivorship at both life stages when compared with the offspring of outcrossed hermaphrodites or females. In one population the fitness of offspring produced by within‐population outcrossing of females was significantly less than the fitness of offspring produced by crossing females with hermaphrodites from other populations. Self‐fertilization of hermaphrodites produced a smaller proportion of hermaphroditic offspring than did outcrossing hermaphrodites. Outcrossing females within populations produced a smaller proportion of hermaphrodite offspring than did crossing females with hermaphrodites from other populations. These results are consistent with a cytonuclear system of sex determination with dominant nuclear restorers, and are discussed with regard to how the mating system and the genetics of sex determination interact to influence the evolution of inbreeding depression.     

The effect of pollination mode on seed performance of Gentiana lutea: a laboratory evaluation of seed germinability

Gentiana lutea L. (yellow gentian, Gentianaceae) is a protected orophyte of central and southern Europe, mainly threatened by the uncontrolled collection of its rhizome, used in traditional medicine and for liquor production. The species is self‐compatible, but outcrossing mediated by pollinators is needed to obtain a viable progeny. In this study, we considered five natural populations belonging to the four subspecies of G. lutea. We performed controlled pollinations in the field (self‐ versus cross‐pollination) followed by seed germination tests in laboratory conditions, adding a solution of gibberellic acid, in order to evaluate the seed performance. A cumulative index of inbreeding depression was calculated considering maternal reproductive output as well as seed performance traits. Seed weight and seed germination performance was similar between seeds resulting from naturally pollinated and pollen‐augmented flowers and higher compared to selfed flowers, highlighting a disadvantage of selfing and the importance of cross‐pollen transfer in natural conditions. However, in the small and isolated population of G. lutea subsp. symphyandra on Mt Grande we found a general reduction in seed germination rate, likely due to increased selfing or mating among close relatives as a consequence of a severe bottleneck. We discuss our results with regards to implications for conservation practices.     

Floral display and habitat fragmentation: Effects on the reproductive success of the threatened mass‐flowering Conospermum undulatum (Proteaceae)

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Reproductive ecology of Thymelaea velutina (Thymelaeaceae)- Factors contributing to the maintenance of heterocarpy

Thymelaea velutina (Thymelaeaceae) is a dioecious shrub that presents a unique type of heterocarpy which consists of the simultaneous production of dry and fleshy fruits. It is endemic to the Balearic Islands (Western Mediterranean) and is found both in dunes and mountain areas. The goal of this study was to identify which factors influence the production of both fruit types, examining the variation of their effects at a spatio-temporal scale (comparing two localities in different years). Specifically, we investigated (1) whether pollen limitation influences the type of fruit produced, and (2) the possible differences in seed size, mass, dispersal capacity, seed predation, germination patterns and seedling survival between fruit types. We also examined if the production of fleshy fruits was modified with the application of gibberellins to reproductive branches. Although fleshy fruits were consistently more abundant than dry ones at both populations, their proportion was significantly higher at the site with greater precipitation. The addition of either pollen or gibberellins did not affect the proportion of each fruit type. Seeds in fleshy fruits are consistently larger, heavier and more likely to be dispersed than seeds in dry fruits, but germinability, germination rate and seedling survival was similar among fruit types. Heterocarpy in this species is currently maintained as there is no apparent factor that exerts any strong selective pressure on either fruit type. The two fruit types might even have different `functions', one serving especially for dispersal and population expansion and the other for producing a seed bank that ensures an eventual germination.     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.