Rapid radiation of angraecoids (Orchidaceae, Angraecinae) in tropical Africa characterised by multiple karyotypic shifts under major environmental instability.

Angraecoid orchids present a remarkable diversity of chromosome numbers, which makes them a highly suitable system for exploring the impact of karyotypic changes on cladogenesis, diversification and morphological differentiation. We compiled an annotated cytotaxonomic checklist for 126 species of Angraecinae, which was utilised to reconstruct chromosomal evolution using a newly-produced, near-comprehensive phylogenetic tree that includes 245 angraecoid taxa. In tandem with this improved phylogenetic framework, using combined Bayesian, maximum likelihood and parsimony approaches on ITS-1 and five plastid markers, we propose a new cladistic nomenclature for the angraecoids, and we estimate a new timeframe for angraecoid radiation based on a secondary calibration, and calculate diversification rates using a Bayesian approach. Coincident divergence dates between clades with identical geographical distributions in the angraecoids and the pantropical orchid genus Bulbophyllum suggest that the same events may have intervened in the dispersal of these two epiphytic groups between Asia, continental Africa, Madagascar and the Neotropics. The major angraecoid lineages probably began to differentiate in the Middle Miocene, and most genera and species emerged respectively around the Late Miocene-Pliocene boundary and the Pleistocene. Ancestral state reconstruction using maximum likelihood estimation revealed an eventful karyotypic history dominated by descending dysploidy. Karyotypic shifts seem to have paralleled cladogenesis in continental tropical Africa, where approximately 90% of the species have descended from at least one inferred transition from n = 17-18 to n = 25 during the Middle Miocene Climatic Transition, followed by some clade-specific descending and ascending dysploidy from the Late Miocene to the Pleistocene. Conversely, detected polyploidy is restricted to a few species lineages mostly originating during the Pleistocene. No increases in net diversification could be related to chromosome number changes, and the apparent net diversification was found to be highest in Madagascar, where karyotypic stasis predominates. Finally, shifts in chromosome number appear to have paralleled the evolution of rostellum structure, leaflessness, and conspicuous changes in floral colour.

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.

Evolution in the Amphi-Atlantic tropical genus Guibourtia (Fabaceae, Detarioideae), combining NGS phylogeny and morphology.

Tropical rain forests support a remarkable diversity of tree species, questioning how and when this diversity arose. The genus Guibourtia (Fabaceae, Detarioideae), characterized by two South American and 13 African tree species growing in various tropical biomes, is an interesting model to address the role of biogeographic processes and adaptation to contrasted environments on species diversification. Combining whole plastid genome sequencing and morphological characters analysis, we studied the timing of speciation and diversification processes in Guibourtia through molecular dating and ancestral habitats reconstruction. All species except G. demeusei and G. copallifera appear monophyletic. Dispersal from Africa to America across the Atlantic Ocean is the most plausible hypothesis to explain the occurrence of Neotropical Guibourtia species, which diverged ca. 11.8 Ma from their closest African relatives. The diversification of the three main clades of African Guibourtia is concomitant to Miocene global climate changes, highlighting pre-Quaternary speciation events. These clades differ by their reproductive characters, which validates the three subgenera previously described: Pseudocopaiva, Guibourtia and Gorskia. Within most monophyletic species, plastid lineages start diverging from each other during the Pliocene or early Pleistocene, suggesting that these species already arose during this period. The multiple transitions between rain forests and dry forests/savannahs inferred here through the plastid phylogeny in each Guibourtia subgenus address thus new questions about the role of phylogenetic relationships in shaping ecological niche and morphological similarity among taxa.

Pleistocene population expansions of shade-tolerant trees indicate fragmentation of the African rainforest during the Ice Ages.

The fossil record in tropical Africa suggests that dry conditions during the Ice Ages caused expansion of savannahs and contraction of the rainforest. Forest refugia have been proposed to be located in areas of Central Africa that currently harbour high rates of endemic species. However, to what extent the forest was fragmented remains unknown. Nuclear microsatellites and plastid sequences of 732 trees of two species occurring in the same habitat-mature lowland evergreen rainforests-but with remarkably different dispersal capacities-animal versus gravity-were analysed. Geographical information system tools revealed intraspecific lineages partially congruent across the two species, suggesting common past barriers to gene flow in Central Africa. According to approximate Bayesian computation, the intraspecific genetic clusters diverged during the Pleistocene (less than 2 Ma), so that intraspecific differentiation is the appropriate scale to test the aridification effect of the Ice Ages on tree populations. Demographic tests revealed clear genetic signals of population expansion in both taxa, possibly following bottleneck events after forest fragmentation, with stronger evidence of expansion after the Penultimate rather than after the Last Glacial Maximum. The differential dispersal capacity may have modulated the particular response of each species to climate change, as revealed by the stronger evidence of expansion found in the animal-dispersed species than in the gravity-dispersed one.

Characterization of microsatellite markers in the African tropical tree species Guibourtia ehie (Fabaceae, Detarioideae).

PREMISE OF THE STUDY: Microsatellite primers (simple sequence repeats [SSRs]) were developed in Guibourtia ehie (Fabaceae, Detarioideae) to study population genetic structure and the history of African vegetation. METHODS AND RESULTS: We isolated 18 polymorphic SSRs from a nonenriched genomic library. This set of primer pairs was tested on four populations, and the results showed two to 16 alleles per locus with mean observed and expected heterozygosities of 0.27 ± 0.05 and 0.57 ± 0.05, respectively. Cross-amplification tests in 13 congeneric species were successful for the four taxa belonging to the subgenus Gorskia. CONCLUSIONS: This set of microsatellite markers will be useful to investigate the phylogeography and population genetics of G. ehie, a key representative of African semideciduous moist forests.

Revealing hidden species diversity in closely related species using nuclear SNPs, SSRs and DNA sequences - a case study in the tree genus Milicia.

BACKGROUND: Species delimitation in closely related plant taxa can be challenging because (i) reproductive barriers are not always congruent with morphological differentiation, (ii) use of plastid sequences might lead to misinterpretation, (iii) rare species might not be sampled. We revisited molecular-based species delimitation in the African genus Milicia, currently divided into M. regia (West Africa) and M. excelsa (from West to East Africa). We used 435 samples collected in West, Central and East Africa. We genotyped SNP and SSR loci to identify genetic clusters, and sequenced two plastid regions (psbA-trnH, trnC-ycf6) and a nuclear gene (At103) to confirm species' divergence and compare species delimitation methods. We also examined whether ecological niche differentiation was congruent with sampled genetic structure. RESULTS: West African M. regia, West African and East African M. excelsa samples constituted three well distinct genetic clusters according to SNPs and SSRs. In Central Africa, two genetic clusters were consistently inferred by both types of markers, while a few scattered samples, sympatric with the preceding clusters but exhibiting leaf traits of M. regia, were grouped with the West African M. regia cluster based on SNPs or formed a distinct cluster based on SSRs. SSR results were confirmed by sequence data from the nuclear region At103 which revealed three distinct 'Fields For Recombination' corresponding to (i) West African M. regia, (ii) Central African samples with leaf traits of M. regia, and (iii) all M. excelsa samples. None of the plastid sequences provide indication of distinct clades of the three species-like units. Niche modelling techniques yielded a significant correlation between niche overlap and genetic distance. CONCLUSIONS: Our genetic data suggest that three species of Milicia could be recognized. It is surprising that the occurrence of two species in Central Africa was not reported for this well-known timber tree. Globally, our work highlights the importance of collecting samples in a systematic way and the need for combining different nuclear markers when dealing with species complexes. Recognizing cryptic species is particularly crucial for economically exploited species because some hidden taxa might actually be endangered as they are merged with more abundant species.

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.

Development and characterization of microsatellite markers in the African deciduous tree Terminalia superba (Combretaceae).

PREMISE OF THE STUDY: Microsatellites were designed and characterized in the African timber forest tree Terminalia superba (Combretaceae). Due to their high variability, these markers are suitable to investigate gene flow patterns and the structure of genetic diversity. METHODS AND RESULTS: From a genomic library obtained by next-generation sequencing, seven monomorphic and 14 polymorphic microsatellite loci were developed. The polymorphic microsatellites displayed two to 27 alleles (mean 11.4; expected heterozygosity range 0.283-0.940, mean 0.736) in one population from southeastern Cameroon. Genotypes were typical of an outbreeding diploid species, although null alleles explain a significant heterozygote deficit in three loci. Cross-amplification in three congeneric species (T. ivorensis, T. avicennioides, and T. mantaly) failed, suggesting that T. superba is rather divergent. CONCLUSIONS: This set of newly developed microsatellite markers will be useful for assessing the genetic diversity, population structure, and demographic history of T. superba in tropical African forests.

Isolation and partial characterization of mutants with elevated lipid content in Chlorella sorokiniana and Scenedesmus obliquus.

This paper describes the isolation and partial biomass characterization of high triacylglycerol (TAG) mutants of Chlorella sorokiniana and Scenedesmus obliquus, two algal species considered as potential source of biodiesel. Following UV mutagenesis, 2000 Chlorella and 2800 Scenedesmus colonies were screened with a method based on Nile Red fluorescence. Several mutants with high Nile Red fluorescence were selected by this high-throughput method in both species. Growth and biomass parameters of the strongest mutants were analyzed in detail. All of the four Chlorella mutants showed no significant changes in growth rate, cell weight, cell size, protein and chlorophyll contents on a per cell basis. Whereas all contained elevated total lipid and TAG content per unit of dry weight, two of them were also affected for starch metabolism, suggesting a change in biomass/storage carbohydrate composition. Two Scenedesmus mutants showed a 1.5 and 2-fold increased cell weight and larger cells compared to the wild type, which led to a general increase of biomass including total lipid and TAG content on a per cell basis. Such mutants could subsequently be used as commercial oleaginous algae and serve as an alternative to conventional petrol.