Complete chloroplast genome of two nutmeg species, Myristica argentea Warb. 1891 and Myristica fatua Houtt. 1774 (Myristicaceae).

Myristica argentea Warb. 1891 and M. fatua Houtt. 1774 are two South-East Asian food tree species. They are harvested from the wild or cultivated for local uses as a condiment (nutmeg and mace), medicine, and source of wood. In this study, we reconstructed the complete chloroplast (cp) genomes of these two species from whole genome sequencing data using the Illumina NovaSeq platform. The genome sizes of M. argentea and M. fatua were respectively 155,871 base pairs (bp) and 155,898 bp, including 126 genes and an overall GC content of 39.20% in both species. Our study provides useful resources for future evolutionary research and diversity analysis of Myristica species.

Dataset from de novo transcriptome assembly of Myristica fatua leaves using MinION nanopore sequencer.

Myristica fatua is a tropical fruit tree species originating from Indonesia. Very few genomic resources are available for the species. We developed a full-length transcriptome assembly using long-read sequencing (MinION Nanopore technology) and produced 4.3 million reads (3.5 G of bases). The assembled full-length transcript was constructed using the RATTLE program and assembled 21,098 transcripts. The transcript ranged from 201 - 14,174 bp, and N50 was 2,017 bp. The transcripts were annotated with the UNIPROT database using BlastX. The functional annotation was performed using Blast2go software. The 8,445 microsatellite motif-containing contigs were identified. The raw reads are deposited in the ENA (European Nucleotide Archive) with ENA experiment accession number ERX6798613.

Domestication Syndrome in Dacryodes edulis (Burseraceae): Comparison of Morphological and Biochemical Traits between Wild and Cultivated Populations.

For millennia, people have harvested fruits from the wild for their alimentation. Gradually, they have started selecting wild individuals presenting traits of interest, protecting and cultivating them. This was the starting point of their domestication. The passage from a wild to a cultivated status is accompanied by a modification of a number of morphological and genetic traits, commonly known as the domestication syndrome. We studied the domestication syndrome in Dacryodes edulis (G.Don) H.J.Lam (known as 'African plum' or 'safoutier/prunier'), a socio-economically important indigenous fruit tree species in West and Central Africa. We compared wild and cultivated individuals for their sex distribution; flower, fruit and seed morphometric characteristics; seed germination temporal dynamic and fruit lipid composition. We found a higher percentage of male and male-hermaphrodite sexual types in wild populations than in cultivated ones; a lower fruit and seed mass in wild individuals; and similar mean time of germination, oil content and fatty acid composition between wild and cultivated individuals. Our results are interpreted in light of the presence of a domestication syndrome in D. edulis.

Shifting perceptions, preferences and practices in the African fruit trade: the case of African plum (Dacryodes edulis) in different cultural and urbanization contexts in Cameroon.

BACKGROUND: Understanding the perceptions, preferences and management practices associated with intraspecific variability of emblematic African tree crops is critical for their sustainable management. In this paper, we examine how the agrobiodiversity of a fruit tree species native to Central Africa, the African plum tree (Dacryodes edulis), is perceived and managed by Cameroonian cultivators. METHODS: Semi-structured interviews and tree surveys were conducted over four months with 441 African plum tree owners from three different ethnic groups (Bamileke, Bassa, Beti) in urban, peri-urban and rural areas. Questions focused on trees owners' perceptions-including the local nomenclature-preferences and management practices related to African plum trees and their intraspecific agrobiodiversity. RESULTS: Across the three ethnic groups in the study area, more than 300 different local varietal names were recorded. These were mainly based on morphological and organoleptic traits, with two-thirds of the names referring to fruit size, skin color and fruit taste. The same traits were used by tree owners to describe their fruit preferences, but their relative importance in shaping fruit preferences varied among groups. The preferences of urban dwellers from different ethnic groups when purchasing African plum fruit focused on the fruit's taste characteristics, while those of rural dwellers differed among ethnic groups. In rural areas, where African plums are sold and consumed by their growers, the preferences of Bassa consumers reflect quantity (fruit size) over quality (fruit taste or skin color) considerations. These preferences are reflected in the choice of seeds used for planting. Bassa owners sought seeds from trees with large fruits (with 34.8% of Bassa owners giving top priority to this trait as a selection criterion) to a significantly greater extent than Bamileke and Beti owners who prioritized taste and skin color instead. Among tree growers who selectively retained African plum trees in their fields, 44% considered tree productivity as a primary selection criterion. CONCLUSIONS: Findings linking perceptions of and preferences for fruit traits to intraspecific tree diversity, with attention to inter-ethnic and rural-urban differences, will help design locally specific measures to conserve the agrobiodiversity of African plum in the context of its ongoing domestication.

Trees and their seed networks: The social dynamics of urban fruit trees and implications for genetic diversity.

Trees are a traditional component of urban spaces where they provide ecosystem services critical to urban wellbeing. In the Tropics, urban trees' seed origins have rarely been characterized. Yet, understanding the social dynamics linked to tree planting is critical given their influence on the distribution of associated genetic diversity. This study examines elements of these dynamics (seed exchange networks) in an emblematic indigenous fruit tree species from Central Africa, the African plum tree (Dacryodes edulis, Burseraceae), within the urban context of Yaoundé. We further evaluate the consequences of these social dynamics on the distribution of the genetic diversity of the species in the city. Urban trees were planted predominantly using seeds sourced from outside the city, resulting in a level of genetic diversity as high in Yaoundé as in a whole region of production of the species. Debating the different drivers that foster the genetic diversity in planted urban trees, the study argues that cities and urban dwellers can unconsciously act as effective guardians of indigenous tree genetic diversity.

Utility of the Mitochondrial Genome in Plant Taxonomic Studies.

Size, structure, and sequence content lability of plant mitochondrial genome (mtDNA) across species has sharply limited its use in taxonomic studies. Historically, mtDNA variation has been first investigated with RFLPs, while the development of universal primers then allowed studying sequence polymorphisms within short genomic regions (<3 kb). The recent advent of NGS technologies now offers new opportunities by greatly facilitating the assembly of longer mtDNA regions, and even full mitogenomes. Phylogenetic works aiming at comparing signals from different genomic compartments (i.e., nucleus, chloroplast, and mitochondria) have been developed on a few plant lineages, and have been shown especially relevant in groups with contrasted inheritance of organelle genomes. This chapter first reviews the main characteristics of mtDNA and the application offered in taxonomic studies. It then presents tips for best sequencing protocol based on NGS data to be routinely used in mtDNA-based phylogenetic studies.

Fine-scale spatial genetic structure, mating, and gene dispersal patterns in Parkia biglobosa populations with different levels of habitat fragmentation.

PREMISE: A good understanding of genetic variation and gene dispersal in tree populations is crucial for their sustainable management, particularly in a context of rapid environmental changes. West African Sudanian savannahs are being fragmented and degraded, partly due to expansion of crop cultivation and monocultures that reduce tree density and may impact pollinators. The population dynamics of important indigenous trees could also be affected. We investigated the influence of habitat fragmentation on patterns of genetic diversity and gene dispersal of a key Sudanian agroforestry tree species, Parkia biglobosa. METHODS: Using 10 highly polymorphic nuclear microsatellites, we genotyped 2475 samples from reproductive trees, seedlings, and embryos in four tree populations presenting different levels of habitat fragmentation. RESULTS: Parkia biglobosa presented similar high genetic diversity across the four populations studied. Genetic diversity and inbreeding were similar between adults and embryo cohorts. In all four populations, the selfing rate was less than 1%. The effective number of pollen donors per tree was high (NEP ~ 18-22), as was the pollen immigration rate (from 34 to 74%). Pollen dispersal was characterized by a fat-tailed distribution with mean estimates exceeding 200 m. In three populations, stem diameter had a pronounced effect on male reproductive success. Here, the highest male reproductive success was observed in trees with a diameter at breast height between 60 and 75 cm. CONCLUSIONS: At the scale analyzed, fragmentation does not seem to pose limitations to gene flow in any of the sites investigated, regardless of the landscape configuration associated with the different tree stands. The study provides useful insights on the reproductive biology of an important tree species in the West African savannahs.

Microsatellite markers development for Indonesian nutmeg (Myristica fragrans Houtt.) and transferability to other Myristicaceae spp.

Myristica fragrans (Myristicaceae) is a tropical evergreen tree that yields the two famous spices: nutmeg and mace. Despite its socio-economic importance, the spatial distribution of its genetic diversity is barely documented. In this aim, 48 nuclear microsatellite markers were isolated of which 14 were polymorphic in M. fragrans. Number of alleles per locus ranged from 2 to 6. The level of observed heterozygosity ranged from 0.038 to 0.929 across loci. Transferability of these microsatellites in other Myristica species (M. fatua, M. argentea, and M. crassipes) and Myristicaceae species (Horsfieldia palauensis) was tested and successful. These new microsatellites will be useful for future investigation on genetic diversity and population structure of M. fragrans and phylogenetically-related species.

Population genomics of the widespread African savannah trees Afzelia africana and Afzelia quanzensis reveals no significant past fragmentation of their distribution ranges.

PREMISE: Few studies have addressed the evolutionary history of tree species from African savannahs. Afzelia contains economically important timber species, including two species widely distributed in African savannahs: A. africana in the Sudanian region and A. quanzensis in the Zambezian region. We aimed to infer whether these species underwent range fragmentation and/or demographic changes, possibly reflecting how savannahs responded to Quaternary climate changes. METHODS: We characterized the genetic diversity and structure of these species across their distribution ranges using nuclear microsatellites (SSRs) and genotyping-by-sequencing (GBS) markers. Six SSR loci were genotyped in 241 A. africana and 113 A. quanzensis individuals, while 2800 high-quality single nucleotide polymorphisms (SNPs) were identified in 30 A. africana individuals. RESULTS: Both species appeared to be mainly outcrossing. The kinship between individuals decayed with the logarithm of the distance at similar rates across species and markers, leading to relatively small Sp statistics (0.0056 for SSR and 0.0054 for SNP in A. africana, 0.0075 for SSR in A. quanzensis). The patterns were consistent with isolation by distance expectations in the absence of large-scale geographic gradients. Bayesian clustering of SSR genotypes did not detect genetic clusters within species. In contrast, SNP data resolved intraspecific genetic clusters in A. africana, illustrating the higher resolving power of GBS. However, these clusters revealed low levels of differentiation and no clear geographical entities, so that they were interpreted as resulting from the isolation by distance pattern rather than from past population fragmentation. CONCLUSIONS: These results suggest that populations have remained connected throughout the large, continuous savannah landscapes. The absence of clear phylogeographic discontinuities, also found in a few other African savannah trees, indicates that their distribution ranges have not been significantly fragmented during the climatic oscillations of the Pleistocene, in contrast to patterns commonly found in African rainforest trees.

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.

Comparative analysis of two sister Erythrophleum species (Leguminosae) reveal contrasting transcriptome-wide responses to early drought stress.

With the ongoing climate change, African rainforests are expected to experience severe drought events in the future. In Africa, the tropical genus Erythrophleum (Fabaceae) includes two forest sister timber tree species displaying contrasting geographical distributions. Erythrophleum ivorense is adapted to wet evergreen Guineo-Congolian forests, whereas E. suaveolens occurs in a wider range of climates, being found in moist dense forests but also in gallery forests under a relatively drier climate. This geographical distribution pattern suggests that the two species might cope differently to drought at the genomic level. Yet, the genetic basis of tolerance response to drought stress in both species is still uncharacterized. To bridge this gap, we performed an RNA-seq approach on seedlings from each species to monitor their transcriptional responses at different levels of drought stress (0, 2 and 6 weeks after stopping watering seedlings). Monitoring of wilting symptoms revealed that E. suaveolens displayed an earlier phenotypic response to drought stress than E. ivorense. At the transcriptomic level, results revealed 2020 (1204 down-regulated/816 up-regulated) and 1495 differentially expressed genes (DEGs) in response to drought stress from a total of 67,432 and 66,605 contigs assembled in E. ivorense and E. suaveolens, respectively. After identifying 30,374 orthologs between species, we found that only 7 of them were DEGs shared between species, while 587 and 458 were differentially expressed only in E. ivorense or E. suaveolens, respectively. GO and KEGG enrichment analysis revealed that the two species differ in terms of significantly regulated pathways as well as the number and expression profile of DEGs (Up/Down) associated with each pathway in the two stress stages. Our results suggested that the two studied species react differently to drought. E. suaveolens seems displaying a prompt response to drought at its early stage strengthened by the down-regulation of many DEGs encoding for signaling and metabolism-related pathways. A considerable up-regulation of these pathways was also found in E. ivorense at the late stage of drought, suggesting this species may be a late responder. Overall, our data may serve as basis for further understanding the genetic control of drought tolerance in tropical trees and favor the selection of crucial genes for genetically enhancing drought resistance.

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.

Characterization of microsatellite markers in two exploited African trees, Entandrophragma candollei and E. utile (Meliaceae).

PREMISE OF THE STUDY: Multiplexes of nuclear microsatellite primers were developed to investigate population genetic structure and diversity in two exploited African rainforest trees: Entandrophragma candollei and E. utile (Meliaceae). METHODS AND RESULTS: Microsatellite isolation was performed simultaneously on two nonenriched genomic libraries after next-generation sequencing. We developed 16 and 22 polymorphic markers for E. candollei and E. utile in three and four multiplexes, respectively. The number of alleles ranged from two to 17 for E. candollei and from three to 19 for E. utile. Mean expected and observed heterozygosity ranged between 0.75 ± 0.13 and 0.55 ± 0.23 for E. candollei and between 0.73 ± 0.10 and 0.49 ± 0.2 for E. utile. CONCLUSIONS: These sets of nuclear microsatellite markers constitute useful tools for exploring gene flow patterns in these two Entandrophragma species.

Evolution in African tropical trees displaying ploidy-habitat association: The genus Afzelia (Leguminosae).

Polyploidy has rarely been documented in rain forest trees but it has recently been found in African species of the genus Afzelia (Leguminosae), which is composed of four tetraploid rain forest species and two diploid dry forest species. The genus Afzelia thus provides an opportunity to examine how and when polyploidy and habitat shift occurred in Africa, and whether they are associated. In this study, we combined three plastid markers (psbA, trnL, ndhF), two nuclear markers (ribosomal ITS and the single-copy PEPC E7 gene), plastomes (obtained by High Throughput Sequencing) and morphological traits, with an extensive taxonomic and geographic sampling to explore the evolutionary history of Afzelia. Both nuclear DNA and morphological vegetative characters separated diploid from tetraploid lineages. Although the two African diploid species were well differentiated genetically and morphologically, the relationships among the tetraploid species were not resolved. In contrast to the nuclear markers, plastid markers revealed that one of the diploid species forms a well-supported clade with the tetraploids, suggesting historical hybridisation, possibly in relation with genome duplication (polyploidization) and habitat shift from dry to rain forests. Molecular dating based on fossil-anchored gene phylogenies indicates that extant Afzelia started diverging c. 14.5 or 20Ma while extant tetraploid species started diverging c. 7.0 or 9.4Ma according to plastid and nuclear DNA, respectively. Additional studies of tropical polyploid plants are needed to assess whether the ploidy-habitat association observed in African Afzelia would reflect a role of polyploidization in niche divergence in the tropics.

Microsatellite development for the genus Guibourtia (Fabaceae, Caesalpinioideae) reveals diploid and polyploid species.

PREMISE OF THE STUDY: Nuclear microsatellites (nSSRs) were designed for Guibourtia tessmannii (Fabaceae, Caesalpinioideae), a highly exploited African timber tree, to study population genetic structure and gene flow. METHODS AND RESULTS: We developed 16 polymorphic nSSRs from a genomic library tested in three populations of G. tessmannii and two populations of G. coleosperma. These nSSRs display three to 14 alleles per locus (mean 8.94) in G. tessmannii. Cross-amplification tests in nine congeneric species demonstrated that the genus Guibourtia contains diploid and polyploid species. Flow cytometry results combined with nSSR profiles suggest that G. tessmannii is octoploid. CONCLUSIONS: nSSRs revealed that African Guibourtia species include both diploid and polyploid species. These markers will provide information on the mating system, patterns of gene flow, and genetic structure of African Guibourtia species.

Mitochondrial genome and plant taxonomy.

The lability in size, structure, and sequence content of mitochondrial genome (mtDNA) across plant species has sharply limited its use in taxonomic studies. However, due to the new opportunities offered by the availability of complete mtDNA sequence in plant species and the subsequent development of universal primers, the number of mtDNA-based molecular studies has recently increased. Historically, universal primers have enabled to characterize mtDNA polymorphism mainly by the RFLP technique. This methodology has been progressively replaced by Sanger DNA sequencing, which actually provides the full phylogenetic information content of a DNA fragment (single nucleotide, insertion/deletion, and single sequence repeat length polymorphism). This chapter presents a sequencing working protocol to be routinely used in mtDNA-based phylogenetic studies.

Comparative phylogeography in rainforest trees from Lower Guinea, Africa.

Comparative phylogeography is an effective approach to assess the evolutionary history of biological communities. We used comparative phylogeography in fourteen tree taxa from Lower Guinea (Atlantic Equatorial Africa) to test for congruence with two simple evolutionary scenarios based on physio-climatic features 1) the W-E environmental gradient and 2) the N-S seasonal inversion, which determine climatic and seasonality differences in the region. We sequenced the trnC-ycf6 plastid DNA region using a dual sampling strategy: fourteen taxa with small sample sizes (dataset 1, mean n = 16/taxon), to assess whether a strong general pattern of allele endemism and genetic differentiation emerged; and four taxonomically well-studied species with larger sample sizes (dataset 2, mean n = 109/species) to detect the presence of particular shared phylogeographic patterns. When grouping the samples into two alternative sets of two populations, W and E, vs. N and S, neither dataset exhibited a strong pattern of allelic endemism, suggesting that none of the considered regions consistently harboured older populations. Differentiation in dataset 1 was similarly strong between W and E as between N and S, with 3-5 significant F ST tests out of 14 tests in each scenario. Coalescent simulations indicated that, given the power of the data, this result probably reflects idiosyncratic histories of the taxa, or a weak common differentiation pattern (possibly with population substructure) undetectable across taxa in dataset 1. Dataset 2 identified a common genetic break separating the northern and southern populations of Greenwayodendron suaveolens subsp. suaveolens var. suaveolens, Milicia excelsa, Symphonia globulifera and Trichoscypha acuminata in Lower Guinea, in agreement with differentiation across the N-S seasonal inversion. Our work suggests that currently recognized tree taxa or suspected species complexes can contain strongly differentiated genetic lineages, which could lead to misinterpretation of phylogeographic patterns. Therefore the evolutionary processes of such taxa require further study in African tropical rainforests.

Large-scale pattern of genetic differentiation within African rainforest trees: insights on the roles of ecological gradients and past climate changes on the evolution of Erythrophleum spp (Fabaceae).

BACKGROUND: The evolutionary events that have shaped biodiversity patterns in the African rainforests are still poorly documented. Past forest fragmentation and ecological gradients have been advocated as important drivers of genetic differentiation but their respective roles remain unclear. Using nuclear microsatellites (nSSRs) and chloroplast non-coding sequences (pDNA), we characterised the spatial genetic structure of Erythrophleum (Fabaceae) forest trees in West and Central Africa (Guinea Region, GR). This widespread genus displays a wide ecological amplitude and taxonomists recognize two forest tree species, E. ivorense and E. suaveolens, which are difficult to distinguish in the field and often confused. RESULTS: Bayesian-clustering applied on nSSRs of a blind sample of 648 specimens identified three major gene pools showing no or very limited introgression. They present parapatric distributions correlated to rainfall gradients and forest types. One gene pool is restricted to coastal evergreen forests and corresponds to E. ivorense; a second one is found in gallery forests from the dry forest zone of West Africa and North-West Cameroon and corresponds to West-African E. suaveolens; the third gene pool occurs in semi-evergreen forests and corresponds to Central African E. suaveolens. These gene pools have mostly unique pDNA haplotypes but they do not form reciprocally monophyletic clades. Nevertheless, pDNA molecular dating indicates that the divergence between E. ivorense and Central African E. suaveolens predates the Pleistocene. Further Bayesian-clustering applied within each major gene pool identified diffuse genetic discontinuities (minor gene pools displaying substantial introgression) at a latitude between 0 and 2°N in Central Africa for both species, and at a longitude between 5° and 8°E for E. ivorense. Moreover, we detected evidence of past population declines which are consistent with historical habitat fragmentation induced by Pleistocene climate changes. CONCLUSIONS: Overall, deep genetic differentiation (major gene pools) follows ecological gradients that may be at the origin of speciation, while diffuse differentiation (minor gene pools) are tentatively interpreted as the signature of past forest fragmentation induced by past climate changes.

How effective are DNA barcodes in the identification of African rainforest trees?

BACKGROUND: DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species. METHODOLOGY/PRINCIPAL FINDINGS: We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95-100% success), but less for species identification (71-88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84-90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA. CONCLUSIONS/SIGNIFICANCE: Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications.

Characterization of new microsatellite loci isolated from Santiria trimera (Burseraceae).

PREMISE OF THE STUDY: To study the genetic structure among three morphotypes of an African rainforest tree species, Santiria trimera, nuclear microsatellite markers were isolated and characterized. METHODS AND RESULTS: Seven polymorphic loci were isolated using a pyrosequencing-based protocol and successfully amplified on three different morphotypes of S. trimera. For six of the seven loci, there is at least one private allele for one of the three morphotypes. The mean effective number of alleles is about four for each of the three morphotypes. CONCLUSIONS: These microsatellite markers are promising to explore the genetic delimitation among sympatric morphotypes found in Gabonese forests and to study the spatial genetic structure within each gene pool.