Development and characterization of microsatellite markers in the African timber tree species Cylicodiscus gabunensis (Fabaceae).

BACKGROUND: Cylicodiscus gabunensis (Fabaceae) or 'Okan' is a Central African multipurpose timber species that is used for heavy construction and traditional medicine. Despite being currently heavily exploited, the species shows a low population density and a natural regeneration deficit in dense forest. METHODS AND RESULTS: We aimed to characterize polymorphic microsatellite markers that can be used to study patterns of genetic structure and gene flow (mating system, pollen and seed dispersal and inbreeding depression) and ultimately, help to develop sustainable forest management practices. We developed 24 polymorphic markers that can be amplified in three PCR multiplexes that were tested in 647 individuals of C. gabunensis from three populations. The number of alleles ranged from 4 to 27 and the average observed and expected heterozygosity across loci and populations were Ho = 0.585 (0.081-0.936) and He = 0.510 (0.076-0.914), respectively. CONCLUSIONS: This set of markers is a useful tool for exploring intra-specific diversity, genetic structure and gene flow of C. gabunensis.

Seed and pollen dispersal distances in two African legume timber trees and their reproductive potential under selective logging.

The natural regeneration of tree species depends on seed and pollen dispersal. To assess whether limited dispersal could be critical for the sustainability of selective logging practices, we performed parentage analyses in two Central African legume canopy species displaying contrasted floral and fruit traits: Distemonanthus benthamianus and Erythrophleum suaveolens. We also developed new tools linking forward dispersal kernels with backward migration rates to better characterize long-distance dispersal. Much longer pollen dispersal in D. benthamianus (mean distance dp  = 700 m, mp  = 52% immigration rate in 6 km2 plot, s = 7% selfing rate) than in E. suaveolens (dp  = 294 m, mp  = 22% in 2 km2 plot, s = 20%) might reflect different insect pollinators. At a local scale, secondary seed dispersal by vertebrates led to larger seed dispersal distances in the barochorous E. suaveolens (ds  = 175 m) than in the wind-dispersed D. benthamianus (ds  = 71 m). Yet, seed dispersal appeared much more fat-tailed in the latter species (15%-25% seeds dispersing >500 m), putatively due to storm winds (papery pods). The reproductive success was correlated to trunk diameter in E. suaveolens and crown dominance in D. benthamianus. Contrary to D. benthamianus, E. suaveolens underwent significant assortative mating, increasing further the already high inbreeding of its juveniles due to selfing, which seems offset by strong inbreeding depression. To achieve sustainable exploitation, seed and pollen dispersal distances did not appear limiting, but the natural regeneration of E. suaveolens might become insufficient if all trees above the minimum legal cutting diameter were exploited. This highlights the importance of assessing the diameter structure of reproductive trees for logged species.

Isolation of microsatellite loci in the African tree species Staudtia kamerunensis (Myristicaceae) using high-throughput sequencing.

Staudtia kamerunensis (Myristicaceae) or 'Niové' is an evergreen tree widespread in Central African moist forests. The bark and seeds are used in traditional medicine, yet the tree is mainly harvested for its high quality, multi-purpose timber. To facilitate sustainable harvesting and conservation of the species, we aim to develop microsatellite markers that can be used to study the mating system, gene flow, genetic diversity and population structure. Genomic DNA of S. kamerunensis was sequenced on an Illumina MiSeq platform, generating 195,720 paired-end reads with 3671 sequences containing microsatellites. Amplification tests resulted in the development of 16 highly polymorphic microsatellite loci of which 14 were tested in 183 individuals of S. kamerunensis from three populations. The number of detected alleles per locus ranged from 15 to 39 and the average observed and expected heterozygosity across loci and populations were Ho = 0.713 (0.14-0.97) and He = 0.879 (0.19-0.95) respectively. The high levels of polymorphism observed in the newly developed microsatellite markers demonstrate their usefulness to study gene flow, population structure and spatial distribution of genetic diversity in S. kamerunensis.

Extensive seed and pollen dispersal and assortative mating in the rain forest tree Entandrophragma cylindricum (Meliaceae) inferred from indirect and direct analyses.

Pollen and seed dispersal are key processes affecting the demographic and evolutionary dynamics of plant species and are also important considerations for the sustainable management of timber trees. Through direct and indirect genetic analyses, we studied the mating system and the extent of pollen and seed dispersal in an economically important timber species, Entandrophragma cylindricum (Meliaceae). We genotyped adult trees, seeds and saplings from a 400-ha study plot in a natural forest from East Cameroon using eight nuclear microsatellite markers. The species is mainly outcrossed (t = 0.92), but seeds from the same fruit are often pollinated by the same father (correlated paternity, rp  = 0.77). An average of 4.76 effective pollen donors (Nep ) per seed tree contributes to the pollination. Seed dispersal was as extensive as pollen dispersal, with a mean dispersal distance in the study plot approaching 600 m, and immigration rates from outside the plot to the central part of the plot reaching 40% for both pollen and seeds. Extensive pollen- and seed-mediated gene flow is further supported by the weak, fine-scale spatial genetic structure (Sp statistic = 0.0058), corresponding to historical gene dispersal distances (σg ) reaching approximately 1,500 m. Using an original approach, we showed that the relatedness between mating individuals (Fij  = 0.06) was higher than expected by chance, given the extent of pollen dispersal distances (expected Fij  = 0.02 according to simulations). This remarkable pattern of assortative mating could be a phenomenon of potentially consequential evolutionary and management significance that deserves to be studied in other plant populations.

Fluorescent dye particles as pollen analogues for measuring pollen dispersal in an insect-pollinated forest herb.

In flowering plants, pollen dispersal is often the major contributing component to gene flow, hence a key parameter in conservation genetics and population biology. A cost-effective method to assess pollen dispersal consists of monitoring the dispersal of fluorescent dyes used as pollen analogues. However, few comparisons between dye dispersal and realized pollen dispersal have been performed to validate the method. We investigated pollen dispersal in two small populations of the insect-pollinated herb Primula elatior from urban forest fragments using direct (paternity analyses based on microsatellite DNA markers) and indirect (fluorescent dyes) methods. We compared these methods using two approaches, testing for the difference between the distance distributions of observed dispersal events and estimating parameters of a dispersal model, and related these results to dye dispersal patterns in three large populations. Dye and realized (based on paternity inference) pollen dispersal showed exponential decay distributions, with 74.2-94.8% of the depositions occurring at <50 m and a few longer distance dispersal events (up to 151 m). No significant difference in curve shape was found between dye and realized pollen dispersal distributions. The best-fitting parameters characterizing the dye dispersal model were consistent with those obtained for realized pollen dispersal. Hence, the fluorescent dye method may be considered as reliable to infer realized pollen dispersal for forest herbs such as P. elatior. However, our simulations reveal that large sample sizes are needed to detect moderate differences between dye and realized pollen dispersal patterns because the estimation of dispersal parameters suffers low precision.

Forest refugia revisited: nSSRs and cpDNA sequences support historical isolation in a wide-spread African tree with high colonization capacity, Milicia excelsa (Moraceae).

The impact of the Pleistocene climate oscillations on the structure of biodiversity in tropical regions remains poorly understood. In this study, the forest refuge theory is examined at the molecular level in Milicia excelsa, a dioecious tree with a continuous range throughout tropical Africa. Eight nuclear microsatellites (nSSRs) and two sequences and one microsatellite from chloroplast DNA (cpDNA) showed a deep divide between samples from Benin and those from Lower Guinea. This suggests that these populations were isolated in separate geographical regions, probably for several glacial cycles of the Pleistocene, and that the nuclear gene pools were not homogenized despite M. excelsa's wind-pollination syndrome. The divide could also be related to seed dispersal patterns, which should be largely determined by the migration behaviour of M. excelsa's main seed disperser, the frugivorous bat Eidolon helvum. Within Lower Guinea, a north-south divide, observed with both marker types despite weak genetic structure (nSSRs: F(ST) = 0.035, cpDNA: G(ST) = 0.506), suggested the existence of separate Pleistocene refugia in Cameroon and the Gabon/Congo region. We inferred a pollen-to-seed dispersal distance ratio of c.1.8, consistent with wide-ranging gene dispersal by both wind and bats. Simulations in an Approximate Bayesian Computation framework suggested low nSSR and cpDNA mutation rates, but imprecise estimates of other demographic parameters, probably due to a substantial gene flow between the Lower Guinean gene pools. The decline of genetic diversity detected in some Gabonese populations could be a consequence of the relatively recent establishment of a closed canopy forest, which could negatively affect M. excelsa's reproductive system.