Inbreeding and small population size reduce seed set in a threatened and fragmented plant species, Lupinus sulphureus ssp. kincaidii (Fabaceae)

Willamette Valley upland prairie in western Oregon, USA, has been reduced to less than 1% of its original historic range following European settlement in the 1850s. Lupinus sulphureus ssp. kincaidii (Kincaid's lupine), a threatened species and the primary larval host plant of the endangered Icaricia icarioides fenderi (Fender's blue butterfly), was historically a panmictic metapopulation. Habitat fragmentation may be causing many of the Kincaid's lupine colonies to display typical symptoms of inbreeding depression, such as low seed production. Hand outcrosses on bagged inflorescences significantly increased seed set and seed fitness compared to open pollination and within-colony pollen treatments. Natural seed set was positively correlated with an increase in the number of Kincaid's lupine patches, suggesting that population size limits seed set. An increase in fruit set was positively correlated with Kincaid's lupine raceme number, raceme density, and the number of lupine patches, demonstrating that floral display and population size increase pollinator service. Restoration of Kincaid's lupine populations should consider measures that lessen the effects of inbreeding depression, especially in small, isolated populations, for the long-term persistence of the species.     

Low levels of genetic variation among southern peripheral populations of the threatened herb, Leontice microrhyncha (Berberidaceae) in Korea

Levels of genetic variation and intrapopulation genetic structures of Leontice microrhyncha S. Moore (Berberidaceae) were assessed for six populations in South Korea, representing the southern most range of a species found in Northeast China and the Korean peninsula. Detected genetic diversity (H_es) was very low (0.024) and F_IS values showed large heterozygote deficiencies. The small percentage of polymorphic loci and numbers of alleles per locus suggest that L. microrhyncha has a history of severe or long-lasting population bottlenecks that have eroded genetic diversity. This study suggests that the Korean population appears to consist of two historically isolated and independently evolving populations. It seems likely that these groups have been isolated and unstable for a significant period of time. However, the effects of recent habitat fragmentation on the historically disjunct and fragmented population system found in L. microrhyncha were not those predicted from the lack of significant relationships between population-level patterns of genetic variation and population sizes. Most non-unique genotypes were shared by most individuals and the lower level of diversity, high levels of inbreeding and population differentiation as well as high rate of seed production indicated that this species is autogamous and self-compatible and probably largely selfing. Therefore, to preserve extant genetic variation, all populations must be protected across the small geographic range of the species to retain both allelic and genotypic diversity.     

Toward the restoration of a sustainable population of a threatened aquatic plant, Nymphoides peltata: Integrated genetic/demographic studies and practices

We provide a brief review of practices and relevant studies for restoration of Nymphoides peltata (yellow floating heart, Asaza), a distylous floating-leaf clonal plant, in Lake Kasumigaura, Japan to develop a model of integrated genetic and demographic conservation for threatened plants. Several theoretically expected issues, such as sudden extinction of small populations with low genetic diversity, limited seed production in small-sized local populations, and significantly high heterozygosity in adults that have survived environmental change, were ascertained through integrated studies on demography with discrimination of genets and genetics using highly polymorphic genetic markers. Investigation of genetic properties of the remnant soil seed bank suggested that the seed bank could potentially restore genetic diversity, although the fitness reduction of seed banks caused by inbreeding could affect the success of restoration. As a result of restoration efforts, increases in the number of local populations and genets in the Lake Kasumigaura metapopulation have led to population recovery.     

The role of floral biology and breeding system on the reproductive success of the narrow endemic Petrocoptis viscosa rothm. (Caryophyllaceae)

Morphological and functional characteristics of flowers may have major effects on their reproductive success. Here, we report a study on the characteristics of flowers of Petrocoptis viscosa, a herb species endemic to the northwest Iberian Peninsula, restricted to crevices in limestone outcrops, and currently occurring in only three populations within an area of less than 30 km². We also investigated the effects of pollen source on indicators of reproductive success. The results show that Petrocoptis viscosa flowers are well adapted to autogamy. Inbreeding depression (as determined by comparison of results obtained after selfing and outcrossing) was negligible for fruit production (δ=−0.05) and mean seed number (δ=−0.11), low for seed germination percentage (δ=0.08), but relatively high for mean seed weight (δ=0.23). The spatial structure and small size of population of Petrocoptis viscosa may mean that inbreeding is frequent in natural habitats. Adaptation of plants to autogamy may therefore be energetically beneficial (i.e. less wasted expenditure on rewards and flowers).     

Reproductive biology and conservation implications of three endangered snapdragon species (Antirrhinum, Plantaginaceae)

About 32% of Antirrhinum species are considered to be endangered; however, no field studies have focused on their reproductive biology. In this work, several aspects of the reproductive biology (flowering phenology, floral biology, breeding system) and potential limits on seed quantity and quality (pollen limitation, inbreeding depression) were studied in natural populations of three endangered species of the genus (Antirrhinum charidemi, Antirrhinum subbaeticum, Antirrhinum valentinum). Results disclose that all three species need insect visitors for seed production since fruit set after autonomous self-pollination was lower than under hand cross-pollination. A. charidemi and A. valentinum were mainly self-incompatible, whereas A. subbaeticum was self-compatible but herkogamous. Supplementary pollination in open-pollinated flowers only increased fruit set and seed set relative to controls in a given population of A. valentinum. Preliminary data on inbreeding depression at early life-cycle stages of the self-compatible A. subbaeticum revealed that the cumulative level was low. Despite the three species being closely related and sharing many ecological characteristics, they show different mating systems, and different factors limit seed quantity and quality. Thus, caution should be taken when making a common conservation plan for a group of closely related taxa.     

Fragmented but not isolated: Contribution of single trees, small patches and long-distance pollen flow to genetic connectivity for Gomortega keule, an endangered Chilean tree

Habitat fragmentation is a major threat to species survival worldwide due to genetic isolation, inbreeding depression, genetic drift and loss of adaptive potential. However the data on how gene-flow changes following habitat fragmentation is contradictory. If there is significant gene-flow between spatially isolated populations then limited conservation resources could be directed away from projects to ‘establish genetic connectivity’ and used to address other consequences of habitat fragmentation.This research focused on an endangered tree species Gomortega keule (Gomortegaceae) in a fragmented landscape in the Central Chile Biodiversity Hotspot and addressed three questions: (1) How far does pollen move between pollen donors and seed trees and what is the shape of the dispersal curve? (2) Do insect pollinators travel outside of forest patches? (3) Do small populations and single trees contribute to genetic connectivity across the landscape?Paternity analysis results show that G. keule’s insect pollinators travel outside of forest patches, over distances of 6 km, beyond the scale of population fragmentation or genetic structure. Pollen moved from small sites and single trees into large sites, as well as in the other direction, indicating these sites play a key role as functioning elements of the wider population and as stepping stones between sites. Fragmentation at the scale investigated has not led to genetic isolation, thus genetic connectivity per se is not a conservation priority. Other consequences of land-use change, specifically continuing habitat loss and population reduction, still threaten the survival of the species.     

The role of genetic mechanisms of sex determination in the survival of small populations of Silene littorea: A reintroduction experiment

Loss of genetic variation in populations may have other effects than inbreeding depression and loss of adaptive potential. In the case of the numerous gynodioecious plant species having cytonuclear systems of sex determination with dominant nuclear restorers of the male function, reduced genetic variation and increased inbreeding would increase the proportion of non-restorer recessive genes found in homozygosity, and therefore that of female plants producing pistillate flowers. This could have opposite effects on the extinction risk of the population. Female plants produce no pollen and may cause pollen limitation, but at the same time they may reduce average homozygosity and inbreeding depression because they are forced outcrossers. We observed that inbreeding indeed increased the frequency of female plants and pistillate flowers in the population, and that this effect was accompanied by reductions in population recruitment independent of inbreeding depression and likely due to pollen limitation. These results suggest that dominant nuclear restorers of pollen production speed up the extinction of small populations of Silene littorea, and that sex determination mechanisms might be an important factor to consider in the conservation of many plant species.     

Boron increases pollen quality,pollination, and fertility of different genetic lines of pepper

Using different genetic lines of pepper, this study analyses the effect of boron (B) on pollen quality and its capacity to pollinate and fertilize. Three different B concentrations (100, 150, and 200 mg L^?1) were applied to six genetic lines, evaluating the in vitro pollen quality and pollen formation. In a second experiment, the highest and the lowest B concentrations were applied to two low-pollen genetic lines, evaluating the growth of the pollen tubes in flowers pollinated with treated pollen. The higher B concentrations significantly improved pollen quality and formation in vitro, while improved pollination and fecundity was observed after monitoring the pollen tubes. For all variables, the specific genetic line also showed some effect. This technique represents a tool for improving pollen in manual hybrid seed programs.     

Seed production, germinability and seedling growth for a bird-pollinated shrub in fragments of kwongan in south-west Australia

In this study we investigate the effect of population size on the proportion of flowers that produce a fruit (fruit set), the number of seeds per fruit (seed set), seed germinability, seedling mortality and growth in a range of population fragments for the bird-pollinated mixed mating system shrub Calothamnus quadrifidus R. Br. (Myrtaceae). We found no significant linear relationship (p < 0.05) between population fragment size and fruit set in any of the three years reproduction was studied. In contrast, we found a very strong positive correlation between the number of seeds produced per fruit and increasing population fragment size for each of the three years. We found no significant linear relationships between population fragment size and seed germination, or seedling growth and mortality. The most plausible explanation for the decline in seed set is increased inbreeding in smaller populations. Although a previous mating system analysis with allozymes did not reflect the above, we present evidence from other lines of inquiry to indicate that inbreeding does increase in smaller populations, but is masked by post-zygotic lethal systems that eliminate genetically incompetent homozygous embryos. We found no evidence that highly mobile pollinators transporting pollen among fragments rescue small fragments from inbreeding. We discuss the implications of our findings for the conservation of plant diversity in fragments of species rich Mediterranean climate shrublands.     

Genetic consequences of using seed mixtures in restoration: A case study of a wetland plant Lychnis flos-cuculi

Sowing with seed mixtures is a common practice in restoring species-rich communities in areas with impoverished species pools. The potential genetic consequences of using these mixtures, however, are poorly understood and often not considered in practical restoration. We investigated genetic diversity, inbreeding and genetic structure of samples collected from 26 populations of the common wetland plant species Lychnis flos-cuculi in an agricultural region in Switzerland. Some of these populations were natural, while others had been sown several years ago. This enabled us to compare the genetic composition of populations of indigenous origin with those originating from commercially produced seed mixtures. Gene diversity and allelic richness were similar in natural and sown populations. In contrast, inbreeding coefficients were three times higher in sown than in natural populations. The sown populations were genetically distinct from the native populations. We distinguished two homogeneous gene pools that presumably originated from different source populations used to produce seed mixtures. The use of commercially produced seeds may alter the genetic diversity and structure of plant populations. The observed higher inbreeding coefficients of sown populations could lead to reduced population viability. To restore genetically diverse populations, the seeds for further propagation should be collected from numerous individuals in large and non-isolated populations nearby restored sites. Ex situ stocks for the production of commercial seed mixtures should only be propagated for a few generations to avoid negative effects such as inbreeding or loss of local adaptation.     

Pollen transfer from invasive Carpobrotus spp. to natives - A study of pollinator behaviour and reproduction success

The influence of invasive plant species on native community composition is well-documented, but less is known about underlying mechanisms. Especially scarce is knowledge about effects on biotic interactions such as relationships between native plants and their pollinators. In this study we investigate if pollen transfer from the invasive and highly pollen productive Carpobrotus spp. affects seed production and/or seed quality in three native species. We monitored pollinator movements and pollen loads on pollinators and native stigmas, and in a field pollination experiment we investigated the effect of invasive pollen on reproduction. Invasive pollen adhered to pollinators, pollinators switched from Carpobrotus spp. to natives, invasive pollen was transferred to native stigmas, and it affected seed production in one species. Although all possible steps for interference with seed production were found to be qualitatively taken, invasive pollen has probably little impact on the native community because the frequency of invasive pollen transfer to natives was low. However, pollination interactions may change with plant abundance and our study provides evidence that pollen transfer from Carpobrotus spp. to natives does occur and have the potential to affect seed production. We found the species identity of shared pollinators to be of importance, higher flower constancy and lower capacity of pollen adherence are likely to result in less invasive pollen transfer.     

Effects of pollination distance on reproduction and offspring performance in Hypochoeris radicata: Experiments with plants from three European regions

Fragmentation of habitats has resulted in increased inbreeding for many plant species, while the introduction of foreign seed material for ecological restoration has resulted in crosses between plants from distant populations. Both processes may reduce plant fitness and increase the risk of extinction. Variation in the expression of inbreeding and outbreeding depression has been found among different genotypes and among populations, but little is known about large scale geographical patterns within species. We studied the effects of cross-proximity on seed production and offspring performance in the perennial meadow plant Hypochoeris radicata (Asteraceae) from five populations in each of three European regions (Bohemia in northwest Czechia, Northern Hesse in central Germany, and Salland in the central Netherlands). Five artificial cross types were conducted with varying proximity of mates: selfing (self), within family crosses (WF), within population crosses (WP), between population crosses (BP), and between region crosses (BR), and the offspring were grown in a common garden. Independent of the region of origin of the maternal plant, selfing, WF and BP crosses resulted in lower seed set and germination than WP crosses, indicating partial self-incompatibility, inbreeding depression and reduced performance in the F1 progeny resulting from outbreeding. However, crosses between regions resulted in similar seed set and germination as within population crosses. For late traits, the effects of inbreeding and interpopulation crosses differed among regions. WP crosses exhibited the highest survival, flowering and multiplicative fitness only in progeny from Czech maternal plants. Our results suggest that the sensitivity of populations to introgression may vary among regions and that outbreeding depression does not necessarily increase with interpopulation distance. However, the current study investigated only effects in the F1 in a common garden and outbreeding depression may be stronger in the F2 and in field populations.     

Pollen-limited production of viable seeds in an endemic dwarf birch, Betula apoiensis, and incomplete reproductive barriers to a sympatric congener, B. ermanii

An endemic dwarf birch, Betula apoiensis, is critically endangered, and two populations of this species are restricted to the ridges of Mt. Apoi in Hokkaido, Japan. We observed the flowering phenology, pollen dispersal, and viable seed production and conducted pollination experiments in order to examine pollen limitation and hybridization with a sympatric congener, B. ermanii. B. apoiensis flowered earlier than B. ermanii but had a more variable flowering time among trees than B. ermanii. The female flowering of B. apoiensis temporally overlapped with the male flowering of B. ermanii as well as with that of B. apoiensis. Pollination experiments demonstrated that seed set and seed germination were higher in female flowers outcrossed than in those that were non-pollinated, selfed, hybridized with B. ermanii pollen, or pollinated naturally. A few selfed or hybrid seeds were filled and germinated, which indicates that self-incompatibility and reproductive barriers are not complete. Logistic regressions of local density of conspecific trees on natural seed set and seed germination were significantly positive. These results suggest that B. apoiensis is pollen-limited.     

Effects of fragmentation on pollinator assemblage, pollen limitation and seed production of Mediterranean myrtle (Myrtus communis)

Pollinator assemblages may shift as a consequence of the destruction and fragmentation of natural habitats. The scarcity of mates and pollinators can lead plant populations to suffer from pollen limitation and a decrease in reproductive performance within fragmented areas. We studied the shift in pollinator assemblages along with pollen limitation and seed production patterns in the Mediterranean shrub Myrtus communis. Our study included six populations contrasting in patch and population size (Large vs. Small) within a fragmented landscape characterized by ∼1% of potential forest coverage. The breeding system in Myrtus communis was self-compatible, but compared with natural pollination, fruit set increased with pollen addition (quantity limited), and seed set (brood size) increased with outcross pollen addition (quality limited). While the pollinator assemblage in Large patches was taxonomically diverse, it was almost monopolized by honeybees in Small patches, where visitation rates were highest and wild bee species were almost absent. In general, Small populations were less pollen limited for fruit set than Large populations, particularly those that received the highest rates of honeybee visits. However, despite differences in fragmentation and pollinators between Large and Small populations, seed production patterns (brood size and seed mass) were rather similar among them, in agreement with similar pollen limitation levels found for brood size. A higher susceptibility of native pollinators to the presence of honeybee hives was found in Small patches, suggesting that the pollinator assemblage may be severely altered when fragmentation occurs in combination with beekeeping. We discuss its implications and effects on plant reproduction in fragmented areas.     

Ecological and genetic effects on demographic processes: pollination, clonality and seed production in Dithyrea maritima

The relative importance of ecological and genetic factors in regulating the demographic processes of small populations is unknown for most rare plants. Dithyrea maritima (beach spectacle pod; Brassicaceae) is a rare, rhizomatously spreading, and self-incompatible herbaceous perennial endemic to coastal sand dunes of southern California. Low seed production in this species may be caused by insufficient pollinator visits, which may be exacerbated by competition for pollinator visits from non-native plants that have invaded the coastal dunes. I tested this ‘ecological’ hypothesis by removing flowers of Cakile maritima and Carpobrotus spp. from three experimental plots on San Nicolas Island. A second possible limitation on seed set is low diversity of genets within subpopulations, which could lead to scarcity of compatible mates. I tested this ‘genetic’ hypothesis by assigning ramets to clones using isozymes, and by relating clonal diversity within populations to seed set. Removal of non-native plants had no detectable effect on pollination service to D. maritima or on seed set, and seed set was unrelated to natural variation in pollination service among populations within a single year. In contrast, clonal diversity within a 10 m² patch was a strong predictor of seed set. Hence a genetic factor explains spatial variation in seed set on San Nicolas Island. On larger spatial and temporal scales, however, pollination service also affects patterns of seed set: a shift in pollination from flies in 1998 to bees in 1999 probably caused a corresponding increase in seed set across San Nicolas Island, while extremely low seed set at another site (San Miguel Island) is probably caused by a virtual absence of pollinators there.     

Genetic data confirms field evidence for natural breeding in a wild taro population (Colocasia esculenta) in northern Queensland, Australia

Taro (Colocasia esculenta) is a vegetatively propagated, starchy root crop cultivated in tropical to temperate regions of the world. Wild forms of taro are distributed from India to southern China, Australia and Melanesia. They are widely known wild food or fodder sources, including in Aboriginal Australia, so it is unclear to what extent wild populations have been dispersed by humans, or naturally via insect-borne pollen and seed dispersal by birds or other animals. In Australia, pollinators and seedlings of taro have not been reported, and a key question is whether or not the wild taro there can breed naturally. Here we report field observations of flowering, fruit set, and an insect pollinator (Colocasiomyia, pupal stage), in a historically significant wild taro population at Hopevale in northern Queensland. The observed pupa is congeneric with two pollinating fly species that have a highly specialised, probably coevolutionary, relationship with taro in neighbouring Papua New Guinea. The field observations suggested the possibility of natural breeding at Hopevale. By analyzing microsatellite diversity within the Hopevale taro population, we found high genetic variation overall, indicative of multiple founding individuals. Two sublocations showed low genetic diversity and strongly negative inbreeding coefficients, consistent with predominantly clonal (vegetative) reproduction. A third sublocation showed high genetic diversity and a weakly negative inbreeding coefficient, indicative of sexual reproduction. This difference between sublocations may relate to microenvironmental conditions that favour seedling establishment in some parts of the site. The data constitute the first demonstration that natural breeding and population spread occurs in Australian wild taro.     

Reproductive biology and ecology of selected rare and endangered Oxalis L. (Oxalidaceae) plant species

About twenty-five percent of all southern African Oxalis species are rare/endangered and highly localized, making them especially vulnerable to extinction through inbreeding, low genetic variation, disrupted biological interactions and stochastic events; all consequences of small population sizes. Moreover, Oxalis displays tristyly, which is a rare and specialized sexual system that includes a strong self-incompatibility component between three floral morphs to promote out-crossing within populations. As tristyly requires the availability of plants with different floral morphs as well as effective pollinators for seed production, this breeding system can affect small populations when fully expressed. Factors that may have an effect on rarity in Oxalis were investigated by focusing on the expression of tristyly, levels of natural seed production, clonality and the ecology of eight rare/highly localized Oxalis species. Field experiments revealed that the reproductive success of some Oxalis species may be hampered by tristyly, resulting in extremely low levels of natural seed production. Other species display a more relaxed expression of self-incompatibility, which in combination with the possibility of cross-pollinations provides reproductive assurance regardless of population structure and pollinator availability. Others are rare and endangered, but appear not to be negatively affected by the tristylous breeding system. Most species are limited by their highly specific habitat requirements and are particularly vulnerable to variation in rainfall patterns.     

Cryptic genetic bottlenecks during restoration of an endangered tropical conifer

Forest restoration programmes aim to use material for re-planting that is genetically diverse and not inbred. However, restricted seed sampling, high variance in reproductive output, and the production of inbred seeds that survive in the nursery but not in the wild can lead to forest restoration stock being genetically compromised. The aim of this study was to evaluate whether the reproductive biology of the New Caledonian endemic conifer Araucaria nemorosa makes it susceptible to these genetic problems and to assess whether there is evidence for genetic bottlenecks and elevated inbreeding in nursery stock compared to seedlings and adults from wild source populations. Reproductive output was low with high variance among trees (only 14% of adult trees surveyed produced mature cones, >50% of examined cones had <10 viable seeds). Evidence for an extreme genetic bottleneck was detected in a nursery population established from cones collected from adult trees. A second nursery population established with seed collected from the forest floor showed no evidence of a genetic bottleneck, but was inbred compared to its wild source population. In light of these results, we do not recommend collecting cones directly from A. nemorosa as an efficient means of establishing genetically diverse stock for restoration programmes. Collecting seed from the forest floor is likely to be more effective, but the planting stock may contain a high proportion of inbred individuals. Collecting established wild seedlings already subjected to natural selection is suggested as an alternative method of maximising the diversity captured, whilst minimising sampling effort and proportion of inbred individuals.     

Impact of selective logging on genetic composition and demographic structure of four tropical tree species

Over-exploitation and fragmentation are serious problems for tropical forests. Most sustainable forest management practices avoid clear-cuts and apply selective logging systems focused on a few commercial species. We applied a simulation model to estimate the impact of such selective logging scenarios on the genetic diversity and demography of four tropical tree species from French Guiana. The simulations used data on genetic and demographic composition, growth, phenology and pollen and seed dispersal obtained for Dicorynia guianensis, Sextonia rubra, Symphonia globulifera and Vouacapoua americana at the experimental site in Paracou. Whereas Symphonia globulifera serves as a model for a species with low logging pressure, the other three species represent the most exploited tree species in French Guiana. In simulations with moderate logging, typical for French Guiana, with large cutting diameter (>60 cm diameter) and long cutting cycles (65 years), the two species V. americana and Sextonia rubra were not able to recover their initial stock at the end of the rotation period, with a large decrease in the number of individuals and in basal area. Under a more intensive logging system (cutting diameter >45 cm diameter, cutting cycles of 30 years) that is common practice in the Brazilian Amazon, only Symphonia globulifera showed no negative impact. Generally, the differences between the genetic parameters in the control scenarios without logging and the logging scenarios were surprisingly small. The main reasons for this were the overlapping of generations and the effective dispersal ability of gene vectors in all species, which guarantee relative homogeneity of the genetic structure in different age classes. Nevertheless, decreasing the population size by logging reduced the number of genotypes and caused higher genetic distances between the original population and the population at the end of the logging cycles. Sensitivity analysis showed that genetic changes in the logging scenarios were principally determined by the growth, densities and cutting diameter of each species, and only to a very small extent by the reproductive system including factors such as pollen and seed dispersal and flowering phenology.     

Breeding system and inbreeding depression in the clonal plant species Knautia arvensis (Dipsacaceae): implications for survival in abandoned grassland

Larger clonal sizes observed in abandoned than in managed grassland could result in increased geitonogamy in Knautia arvensis. The species was investigated for self-compatibility, seed set without pollinators, and inbreeding depression in a greenhouse experiment. Seed set was equivalent in hand-selfed and hand-outcrossed plants (c. 53%), indicating full self-compatibility. Seed set was low (2.8%) in caged, unmanipulated flower heads. There was no significant difference in seed weight, germination, and survival between selfed and outcrossed progeny. Juvenile biomass was significantly lower in selfed than in outcrossed progeny (on average 45% lower). Cumulative fitness calculations for germination, survival, and juvenile biomass showed that the mean inbreeding depression was 58% in offspring produced by selfing. The results suggest that in K. arvensis, which is protected against self-pollination within flower heads through protandry, the likely mechanism for selfing is via geitonogamy among flower heads. Geitonogamy among ramets within the larger clones observed in abandoned grassland could reduce offspring viability in K. arvensis since the fitness of selfed offspring was low. Inbreeding depression could therefore add to the recruitment problems experienced by plants in abandoned grassland, both in K. arvensis and probably in other clonal grassland species, depending on their growth structure and breeding system.