Mate-choice for close kin is associated with improved offspring survival in Lodoicea maldivica, the largest-seeded plant in the world.

We studied spatial patterns of kinship in the offspring of the endangered Lodoicea maldivica, a dioecious palm that produces the largest seed of any plant. Previous research has suggested that restricted seed and pollen dispersal in populations resulted in strong spatial genetic structure. We used microsatellites to genotype young plants and their potential parents at four sites across the species' entire natural range. We determined the most likely parents of each young plant based on the spatial separation of each parent pair, their genetic relatedness, and the level of correlated paternity. We identified both parents (43 female, 54 male) for 139 of 493 young plants. Mean distance between parental pairs was 26.8 m. Correlated paternity was low (0.168), indicating that mother trees were often pollinated by several fathers. Parental pairs were more closely related than expected by chance, suggesting outbreeding depression. Our results highlight the apparent strong mate choice for close kin in parent pairs of surviving offspring. We discuss the alternative biological processes that could lead to this, including the potential for break-up of favourable allelic combinations necessary for the development of the palm's very large seed. Management implications include germinating seeds where they naturally fall, using a diverse range of male plants as pollen donors for hand pollination, and protecting the native community of gecko pollinators.

Tropical and subtropical Asia's valued tree species under threat.

Tree diversity in Asia's tropical and subtropical forests is central to nature-based solutions. Species vulnerability to multiple threats, which affect provision of ecosystem services, is poorly understood. We conducted a region-wide, spatially explicit assessment of the vulnerability of 63 socioeconomically important tree species to overexploitation, fire, overgrazing, habitat conversion, and climate change. Trees were selected for assessment from national priority lists, and selections were validated by an expert network representing 20 countries. We used Maxent suitability modeling to predict species distribution ranges, freely accessible spatial data sets to map threat exposures, and functional traits to estimate threat sensitivities. Species-specific vulnerability maps were created as the product of exposure maps and sensitivity estimates. Based on vulnerability to current threats and climate change, we identified priority areas for conservation and restoration. Overall, 74% of the most important areas for conservation of these trees fell outside protected areas, and all species were severely threatened across an average of 47% of their native ranges. The most imminent threats were overexploitation and habitat conversion; populations were severely threatened by these factors in an average of 24% and 16% of their ranges, respectively. Our model predicted limited overall climate change impacts, although some study species were likely to lose over 15% of their habitat by 2050 due to climate change. We pinpointed specific natural areas in Borneo rain forests as hotspots for in situ conservation of forest genetic resources, more than 82% of which fell outside designated protected areas. We also identified degraded areas in Western Ghats, Indochina dry forests, and Sumatran rain forests as hotspots for restoration, where planting or assisted natural regeneration will help conserve these species, and croplands in southern India and Thailand as potentially important agroforestry options. Our results highlight the need for regionally coordinated action for effective conservation and restoration.

Especies de Árboles Valoradas y Amenazadas de Asia Tropical y Subtropical Resumen La diversidad de árboles en los bosques tropicales y subtropicales de Asia es un eje central para las soluciones basadas en la naturaleza. La vulnerabilidad de las especies ante las múltiples amenazas, las cuales afectan el suministro de servicios ambientales, es un tema poco comprendido. Realizamos una evaluación regional espacialmente explícita de la vulnerabilidad de 63 especies de árboles de importancia socioeconómica ante la sobreexplotación, incendios, sobrepastoreo, conversión del hábitat y cambio climático. Los árboles se seleccionaron para su evaluación a partir de listas nacionales de prioridades, y las selecciones fueron validadas por una red de expertos de 20 países. Usamos el modelado de idoneidad Maxent para predecir el rango de distribución de las especies, conjuntos de datos espaciales de libre acceso para mapear la exposición a las amenazas y rasgos funcionales para estimar la susceptibilidad a las amenazas. Con base en la vulnerabilidad a las amenazas actuales y al cambio climático, identificamos las áreas prioritarias para su conservación y restauración. En general, el 74% de las áreas más importantes para la conservación de estos árboles quedó fuera de las áreas protegidas y todas las especies estaban seriamente amenazadas en promedio en el 47% de su distribución nativa. Las amenazas más inminentes fueron la sobreexplotación y la conversión del hábitat; las poblaciones estuvieron seriamente amenazadas por estos factores en promedio en el 24% y 16% de su distribución, respectivamente. Nuestro modelo predijo un impacto general limitado del cambio climático, aunque algunas especies estudiadas tuvieron la probabilidad de perder más del 15% de su hábitat para el 2050 debido a este factor. Identificamos áreas naturales específicas en las selvas de Borneo como puntos calientes para la conservación in situ de los recursos genéticos forestales, más del 82% de los cuales estaban fuera de las áreas protegidas designadas. También identificamos áreas degradadas en los Ghats Occidentales, los bosques secos de Indochina y las selvas de Sumatra como puntos calientes para la restauración, en donde la siembra o la regeneración natural asistida ayudarán a conservar estas especies. Además, identificamos campos de cultivo al sur de India y Tailandia como potenciales opciones importantes de agrosilvicultura. Nuestros resultados resaltan la necesidad de acciones regionales coordinadas para la conservación y restauración efectivas.

Identification of sex-linked markers in the sexually cryptic coco de mer: are males and females produced in equal proportions?

Lodoicea maldivica (coco de mer) is a long-lived dioecious palm in which male and female plants are visually indistinguishable when immature, only becoming sexually dimorphic as adults, which in natural forest can take as much as 50 years. Most adult populations in the Seychelles exhibit biased sex ratios, but it is unknown whether this is due to different proportions of male and female plants being produced or to differential mortality. In this study, we developed sex-linked markers in Lodoicea using ddRAD sequencing, enabling us to reliably determine the gender of immature individuals. We screened 589 immature individuals to explore sex ratios across life stages in Lodoicea. The two sex-specific markers resulted in the amplification of male-specific bands (Lm123977 at 405 bp and Lm435135 at 130 bp). Our study of four sub-populations of Lodoicea on the islands of Praslin and Curieuse revealed that the two sexes were produced in approximately equal numbers, with no significant deviation from a 1:1 ratio before the adult stage. We conclude that sex in Lodoicea is genetically determined, suggesting that Lodoicea has a chromosomal sex determination system in which males are the heterogametic sex (XY) and females are homogametic (XX). We discuss the potential causes for observed biased sex ratios in adult populations, and the implications of our results for the life history, ecology and conservation management of Lodoicea.

Are patterns of fine-scale spatial genetic structure consistent between sites within tropical tree species?

Documenting the scale and intensity of fine-scale spatial genetic structure (FSGS), and the processes that shape it, is relevant to the sustainable management of genetic resources in timber tree species, particularly where logging or fragmentation might disrupt gene flow. In this study we assessed patterns of FSGS in three species of Dipterocarpaceae (Parashorea tomentella, Shorea leprosula and Shorea parvifolia) across four different tropical rain forests in Malaysia using nuclear microsatellite markers. Topographic heterogeneity varied across the sites. We hypothesised that forests with high topographic heterogeneity would display increased FSGS among the adult populations driven by habitat associations. This hypothesis was not supported for S. leprosula and S. parvifolia which displayed little variation in the intensity and scale of FSGS between sites despite substantial variation in topographic heterogeneity. Conversely, the intensity of FSGS for P. tomentella was greater at a more topographically heterogeneous than a homogeneous site, and a significant difference in the overall pattern of FSGS was detected between sites for this species. These results suggest that local patterns of FSGS may in some species be shaped by habitat heterogeneity in addition to limited gene flow by pollen and seed dispersal. Site factors can therefore contribute to the development of FSGS. Confirming consistency in species' FSGS amongst sites is an important step in managing timber tree genetic diversity as it provides confidence that species specific management recommendations based on species reproductive traits can be applied across a species' range. Forest managers should take into account the interaction between reproductive traits and site characteristics, its consequences for maintaining forest genetic resources and how this might influence natural regeneration across species if management is to be sustainable.

Tracing coco de mer's reproductive history: Pollen and nutrient limitations reduce fecundity.

Habitat degradation can reduce or even prevent the reproduction of previously abundant plant species. To develop appropriate management strategies, we need to understand the reasons for reduced recruitment in degraded ecosystems. The dioecious coco de mer palm (Lodoicea maldivica) produces by far the largest seeds of any plant. It is a keystone species in an ancient palm forest that occurs only on two small islands in the Seychelles, yet contemporary rates of seed production are low, especially in fragmented populations. We developed a method to infer the recent reproductive history of female trees from morphological evidence present on their inflorescences. We then applied this method to investigate the effects of habitat disturbance and soil nutrient conditions on flower and fruit production. The 57 female trees in our sample showed a 19.5-fold variation in flower production among individuals over a seven-year period. Only 77.2% of trees bore developing fruits (or had recently shed fruits), with the number per tree ranging from zero to 43. Flower production was positively correlated with concentrations of available soil nitrogen and potassium and did not differ significantly between closed and degraded habitat. Fruiting success was positively correlated with pollen availability, as measured by numbers and distance of neighboring male trees. Fruit set was lower in degraded habitat than in closed forest, while the proportion of abnormal fruits that failed to develop was higher in degraded habitat. Seed size recorded for a large sample of seeds collected by forest wardens varied widely, with fresh weights ranging from 1 to 18 kg. Synthesis: Shortages of both nutrients and pollen appear to limit seed production of Lodoicea in its natural habitat, with these factors affecting different stages of the reproductive process. Flower production varies widely amongst trees, while seed production is especially low in degraded habitat. The size of seeds is also very variable. We discuss the implications of these findings for managing this ecologically and economically important species.

Evaluating realized seed dispersal across fragmented tropical landscapes: a two-fold approach using parentage analysis and the neighbourhood model.

Despite the importance of seed dispersal for survival of plant species in fragmented landscapes, data on seed dispersal at landscape scales remain sparse. Effective seed dispersal among fragments determines recolonization and plant species persistence in such landscapes. We present the first large-scale (216-km2 ) direct estimates of realized seed dispersal of a high-value timber tree (Dysoxylum malabaricum) across an agro-forest landscape in the Western Ghats, India. Based upon an exhaustive inventory of adult trees and a sample of 488 seedlings all genotyped at 10 microsatellite loci, we estimated realized seed dispersal using parentage analysis and the neighbourhood model. Our estimates found that most realized seed dispersal was within 200 m, which is insufficient to effectively bridge the distances between forest patches. We conclude that using mobility of putative animal dispersers can be misleading when estimating tropical tree species vulnerability to habitat fragmentation. This raises serious concerns about the potential of many tropical trees to recolonize isolated forest patches where high-value tree species have already been removed.

Development of 12 polymorphic microsatellite loci for the endangered Seychelles palm Lodoicea maldivica (Arecaceae).

PREMISE OF THE STUDY: The evolutionarily and ecologically distinct coco de mer palm Lodoicea maldivica (Arecaceae) is endemic to two islands in the Seychelles. Before colonization of the islands by man, the endangered palm formed large monodominant stands, but its natural range is now restricted to four main populations and several patches of isolated individuals. Microsatellite markers were designed to investigate the genetic structure of the remaining natural populations of L. maldivica. METHODS AND RESULTS: We developed 12 polymorphic and three monomorphic microsatellite markers for this species, with a mean number of alleles per locus of 13.2 (range 5-21) and expected heterozygosity values ranging from 0.31-0.91 for the polymorphic loci. CONCLUSIONS: These markers enable us to study the patterns of genetic diversity, contemporary seed dispersal, and the fine-scale spatial genetic structure of this important conservation flagship species.

Predicting dispersal of auto-gyrating fruit in tropical trees: a case study from the Dipterocarpaceae.

Seed dispersal governs the distribution of plant propagules in the landscape and hence forms the template on which density-dependent processes act. Dispersal is therefore a vital component of many species coexistence and forest dynamics models and is of applied value in understanding forest regeneration. Research on the processes that facilitate forest regeneration and restoration is given further weight in the context of widespread loss and degradation of tropical forests, and provides impetus to improve estimates of seed dispersal for tropical forest trees. South-East Asian lowland rainforests, which have been subject to severe degradation, are dominated by trees of the Dipterocarpaceae family which constitute over 40% of forest biomass. Dipterocarp dispersal is generally considered to be poor given their large, gyration-dispersed fruits. However, there is wide variability in fruit size and morphology which we hypothesize mechanistically underpins dispersal potential through the lift provided to seeds mediated by the wings. We explored experimentally how the ratio of fruit wing area to mass ("inverse wing loading," IWL) explains variation in seed dispersal kernels among 13 dipterocarp species by releasing fruit from a canopy tower. Horizontal seed dispersal distances increased with IWL, especially at high wind speeds. Seed dispersal of all species was predominantly local, with 90% of seed dispersing <10 m, although maximum dispersal distances varied widely among species. We present a generic seed dispersal model for dipterocarps based on attributes of seed morphology and provide modeled seed dispersal kernels for all dipterocarp species with IWLs of 1-50, representing 75% of species in Borneo.

Genetic connectivity of the moth pollinated tree Glionnetia sericea in a highly fragmented habitat.

Long-distance gene flow is thought to be one prerequisite for the persistence of plant species in fragmented environments. Human influences have led to severe fragmentation of native habitats in the Seychelles islands, with many species surviving only in small and isolated populations. The endangered Seychelles endemic tree Glionnetia sericea is restricted to altitudes between 450 m and 900 m where the native forest vegetation has been largely lost and replaced with exotic invasives over the last 200 years. This study explores the genetic and ecological consequences of population fragmentation in this species by analysing patterns of genetic diversity in a sample of adults, juveniles and seeds, and by using controlled pollination experiments. Our results show no decrease in genetic diversity and no increase in genetic structuring from adult to juvenile cohorts. Despite significant inbreeding in some populations, there is no evidence of higher inbreeding in juvenile cohorts relative to adults. A Bayesian structure analysis and a tentative paternity analysis indicate extensive historical and contemporary gene flow among remnant populations. Pollination experiments and a paternity analysis show that Glionnetia sericea is self-compatible. Nevertheless, outcrossing is present with 7% of mating events resulting from pollen transfer between populations. Artificial pollination provided no evidence for pollen limitation in isolated populations. The highly mobile and specialized hawkmoth pollinators (Agrius convolvuli and Cenophodes tamsi; Sphingidae) appear to promote extensive gene flow, thus mitigating the potential negative ecological and genetic effects of habitat fragmentation in this species. We conclude that contemporary gene flow is sufficient to maintain genetic connectivity in this rare and restricted Seychelles endemic, in contrast to other island endemic tree species with limited contemporary gene flow.

Forest trees in human modified landscapes: ecological and genetic drivers of recruitment failure in Dysoxylum malabaricum (Meliaceae).

Tropical agro-forest landscapes are global priority areas for biodiversity conservation. Little is known about the ability of these landscapes to sustain large late successional forest trees upon which much forest biodiversity depends. These landscapes are subject to fragmentation and additional habitat degradation which may limit tree recruitment and thus compromise numerous ecosystem services including carbon storage and timber production. Dysoxylum malabaricum is a large canopy tree species in the Meliaceae, a family including many important tropical timber trees. This species is found in highly fragmented forest patches within a complex agro-forest landscape of the Western Ghats biodiversity hot spot, South India. In this paper we combined a molecular assessment of inbreeding with ecological and demographic data to explore the multiple threats to recruitment of this tree species. An evaluation of inbreeding, using eleven microsatellite loci in 297 nursery-reared seedlings collected form low and high density forest patches embedded in an agro-forest matrix, shows that mating between related individuals in low density patches leads to reduced seedling performance. By quantifying habitat degradation and tree recruitment within these forest patches we show that increasing canopy openness and the increased abundance of pioneer tree species lead to a general decline in the suitability of forest patches for the recruitment of D. malabaricum. We conclude that elevated inbreeding due to reduced adult tree density coupled with increased degradation of forest patches, limit the recruitment of this rare late successional tree species. Management strategies which maintain canopy cover and enhance local densities of adult trees in agro-forest mosaics will be required to ensure D. malabaricum persists in these landscapes. Our study highlights the need for a holistic understanding of the incipient processes that threaten populations of many important and rare tropical tree species in human dominated agro-forest landscapes.

Landscape composition has limited impact on local genetic structure in mountain clover, Trifolium montanum L.

Semi-dry grasslands in the European Alps have been increasingly fragmented over the last 150 years. Few studies have investigated the implications of landscape configuration for genetic structure and gene flow among remnant habitat patches. Conservation management of semi-dry grassland plants rarely accounts for possible effects of major landscape elements, such as forest patches, as barriers to gene flow and dispersal via seed and pollen, despite their potential importance for biodiversity conservation. Using 1416 individuals from 61 sampling sites across 2 valleys in South-Eastern Switzerland and Amplified fragment length polymorphism (AFLP) fingerprints, we applied a spatial strip and a circle approach to determine the impact of different landscape elements on genetic differentiation in the semi-dry grassland herb Trifolium montanum (mountain clover). Overall genetic differentiation among sampling sites was low (overall F ST = 0.044). Forest area had no effect on gene flow at the landscape scale, but area of semi-dry grassland, the potential habitat of T. montanum, road area, and altitude influenced genetic differentiation among sampling sites. The observed pattern of genetic differentiation suggests that a future increase in forest area, due to land use abandonment, at least in the short term, are unlikely to directly impact patterns of genetic variation in T. montanum.

Extensive contemporary pollen-mediated gene flow in two herb species, Ranunculus bulbosus and Trifolium montanum, along an altitudinal gradient in a meadow landscape.

BACKGROUND AND AIMS: Genetic connectivity between plant populations allows for exchange and dispersal of adaptive genes, which can facilitate plant population persistence particularly in rapidly changing environments. METHODS: Patterns of historic gene flow, flowering phenology and contemporary pollen flow were investigated in two common herbs, Ranunculus bulbosus and Trifolium montanum, along an altitudinal gradient of 1200-1800 m a.s.l. over a distance of 1 km among five alpine meadows in Switzerland. KEY RESULTS: Historic gene flow was extensive, as revealed by Fst values of 0·01 and 0·007 in R. bulbosus and T. montanum, respectively, by similar levels of allelic richness among meadows and by the grouping of all individuals into one genetic cluster. Our data suggest contemporary pollen flow is not limited across altitudes in either species but is more pronounced in T. montanum, as indicated by the differential decay of among-sibships correlated paternity with increasing spatial distance. Flowering phenology among meadows was not a barrier to pollen flow in T. montanum, as the large overlap between meadow pairs was consistent with the extensive pollen flow. The smaller flowering overlap among R. bulbosus meadows might explain the slightly more limited pollen flow detected. CONCLUSIONS: High levels of pollen flow among altitudes in both R. bulbosus and T. montanum should facilitate exchange of genes which may enhance adaptive responses to rapid climate change.

Eleven microsatellite markers for the mountain clover Trifolium montanum (Fabaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Trifolium montanum to investigate pollen dispersal patterns at landscape scale with a pollen-pool analysis (indirect parentage analysis) as well as in an experimental set-up with a direct paternity analysis. METHODS AND RESULTS: Screening 46 microsatellites developed for T. repens yielded four markers usable in T. montanum. Seven additional ones have been developed specifically for the target species using a 454-sequencing approach. All markers were polymorphic, with an allele number ranging from two to 45 based upon 254 individuals sampled from four populations, and an exclusion probability of 0.999. CONCLUSIONS: These markers proved a useful and reliable molecular tool for use in population genetics and parentage studies of this common grassland herb.

Eight microsatellite markers for the bulbous buttercup Ranunculus bulbosus (Ranunculaceae).

PREMISE OF THE STUDY: Pollen dispersal is a key biological process enabling plant populations to maintain genetic connectivity. Direct estimates of pollen dispersal using paternity assignment or correlated paternity estimates require highly variable genetic markers, of which microsatellites are the markers of choice. • METHODS AND RESULTS: Eight species-specific microsatellites have been developed for Ranunculus bulbosus, combining classical enrichment methods with 454 sequencing. These markers have been used in paternity analysis as well as in pollen-pool analyses and proven to be highly polymorphic (seven to 63 alleles in the largest population studied). An excess of homozygotes in six loci indicate the presence of null alleles. • CONCLUSIONS: These markers are the first microsatellites isolated and tested on R. bulbosus and provide a useful tool for population genetic studies in this common grassland herb.

Patterns of genetic variation across altitude in three plant species of semi-dry grasslands.

BACKGROUND: Environmental gradients caused by altitudinal gradients may affect genetic variation within and among plant populations and inbreeding within populations. Populations in the upper range periphery of a species may be important source populations for range shifts to higher altitude in response to climate change. In this study we investigate patterns of population genetic variation at upper peripheral and lower more central altitudes in three common plant species of semi-dry grasslands in montane landscapes. METHODOLOGY/PRINCIPAL FINDINGS: In Briza media, Trifolium montanum and Ranunculus bulbosus genetic diversity, inbreeding and genetic relatedness of individuals within populations and genetic differentiation among populations was characterized using AFLP markers. Populations were sampled in the Swiss Alps at 1800 (upper periphery of the study organisms) and at 1200 m a.s.l. Genetic diversity was not affected by altitude and only in B. media inbreeding was greater at higher altitudes. Genetic differentiation was slightly greater among populations at higher altitudes in B. media and individuals within populations were more related to each other compared to individuals in lower altitude populations. A similar but less strong pattern of differentiation and relatedness was observed in T. montanum, while in R. bulbosus there was no effect of altitude. Estimations of population size and isolation of populations were similar, both at higher and lower altitudes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that altitude does not affect genetic diversity in the grassland species under study. Genetic differentiation of populations increased only slightly at higher elevation, probably due to extensive (historic) gene flow among altitudes. Potentially pre-adapted genes might therefore spread easily across altitudes. Our study indicates that populations at the upper periphery are not genetically depauperate or isolated and thus may be important source populations for migration under climate change.

Ecological implications of a flower size/number trade-off in tropical forest trees.

BACKGROUND: In angiosperms, flower size commonly scales negatively with number. The ecological consequences of this trade-off for tropical trees remain poorly resolved, despite their potential importance for tropical forest conservation. We investigated the flower size number trade-off and its implications for fecundity in a sample of tree species from the Dipterocarpaceae on Borneo. METHODOLOGY/PRINCIPAL FINDINGS: We combined experimental exclusion of pollinators in 11 species, with direct and indirect estimates of contemporary pollen dispersal in two study species and published estimates of pollen dispersal in a further three species to explore the relationship between flower size, pollinator size and mean pollen dispersal distance. Maximum flower production was two orders of magnitude greater in small-flowered than large-flowered species of Dipterocarpaceae. In contrast, fruit production was unrelated to flower size and did not differ significantly among species. Small-flowered species had both smaller-sized pollinators and lower mean pollination success than large-flowered species. Average pollen dispersal distances were lower and frequency of mating between related individuals was higher in a smaller-flowered species than a larger-flowered confamilial. Our synthesis of pollen dispersal estimates across five species of dipterocarp suggests that pollen dispersal scales positively with flower size. CONCLUSIONS AND THEIR SIGNIFICANCE: Trade-offs embedded in the relationship between flower size and pollination success contribute to a reduction in the variance of fecundity among species. It is therefore plausible that these processes could delay competitive exclusion and contribute to maintenance of species coexistence in this ecologically and economically important family of tropical trees. These results have practical implications for tree species conservation and restoration. Seed collection from small-flowered species may be especially vulnerable to cryptic genetic erosion. Our findings also highlight the potential for differential vulnerability of tropical tree species to the deleterious consequences of forest fragmentation.