Kasso Daïnou

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: FST = 0.035, cpDNA: GST = 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.

Globally, functional traits are weak predictors of juvenile tree growth, and we do not know why

Additional co-authors: Herve Jactel, Xuefei Li, Kaoru Kitajima, Julia Koricheva, Cristina Martinez-Garza, Christian Messier, Alain Paquette, Christopher Philipson, Daniel Piotto, Lourens Poorter, Juan M. Posada, Catherine Potvin, Kalle Rainio, Sabrina E. Russo, Mariacarmen Ruiz-Jaen, Michael Scherer-Lorenzen, Campbell O. Webb, S. Joseph Wright, Rakan A. Zahawi, and Andy Hector

Spatial genetic structure in Milicia excelsa (Moraceae) indicates extensive gene dispersal in a low-density wind-pollinated tropical tree

In this study, we analysed spatial genetic structure (SGS) patterns and estimated dispersal distances in Milicia excelsa (Welw.) C.C. Berg (Moraceae), a threatened wind‐pollinated dioecious African tree, with typically low density (∼10 adults/km2). Eight microsatellite markers were used to type 287 individuals in four Cameroonian populations characterized by different habitats and tree densities. Differentiation among populations was very low. Two populations in more open habitat did not display any correlation between genetic relatedness and spatial distance between individuals, whereas significant SGS was detected in two populations situated under continuous forest cover. SGS was weak with a maximum Sp‐statistic of 0.006, a value in the lower quartile of SGS estimates for trees in the literature. Using a stepwise approach with Bayesian clustering methods, we demonstrated that SGS resulted from isolation by distance and not colonization by different gene pools. Indirect estimates of gene dispersal distances ranged from σg = 1 to 7.1 km, one order of magnitude higher than most estimates found in the literature for tropical tree species. This result can largely be explained by life‐history traits of the species. Milicia excelsa exhibits a potentially wide‐ranging wind‐mediated pollen dispersal mechanism as well as very efficient seed dispersal mediated by large frugivorous bats. Estimations of gene flow suggested no major risk of inbreeding because of reduction in population density by exploitation. Different strategy of seed collection may be required for reforestation programmes among populations with different extent of SGS.

Fitness And Genetic Variation Of Viola Calaminaria, An Endemic Metallophyte: Implications Of Population Structure And History

We investigated variations in genetic diversity and plant fitness in a rare endemic metallophyte of calamine soils, Viola calaminaria, in relation to population size, population connectivity and population history in order to evaluate and discuss potential conservation strategies for the species. Mean population genetic diversity (H(s) = 0.25) of V. calaminaria was similar to endemic non-metallophyte taxa. Twenty-one per cent of the genetic variation was partitioned among populations and a low (9%) but significant differentiation was found among geographical regions. Our results did not support the hypothesis that the acquisition of metal tolerance may result in reduced genetic diversity, and suggested that strict metallophytes do not exhibit higher inter-population differentiation resulting from scattered habitats. There were no relationships between population genetic diversity and population size. Significant correlations were found between plant fitness and (i) population size and (ii) connectivity index. Recently-founded populations exhibited the same level of genetic diversity as ancient populations and also possessed higher plant fitness. There was no indication of strong founder effects in recently-established populations. The results suggest that the creation of habitats through human activities could provide new opportunities for conservation of this species.

Phenological patterns in a natural population of a tropical timber tree species, Milicia excelsa (Moraceae): evidence of Isolation By Time and its interaction with feeding strategies of dispersers

PREMISE OF THE STUDY Population genetic structuring over limited timescales is commonly viewed as a consequence of spatial constraints. Indirect approaches have recently revealed reproductive isolation resulting from flowering time (so-called isolation by time, IBT). Since phenological processes can be subject to selection, the persistence of flowering asynchrony may be due to opposing selective pressures during mating, dispersal, and regeneration phases. Our study aimed to investigate phenology, fruit handling by animals, and their interaction in a timber tree species, Milicia excelsa. METHODS We analyzed phenological data collected over 6 years on 69 genotyped trees in a Cameroonian natural rainforest complemented by data from germination trials and field observations of dispersers. KEY RESULTS Initiation of flowering was correlated with variation in temperature and relative humidity, but was also affected by genetic factors: pairwise differences in flowering time between nearby individuals correlated with kinship coefficient, and earliness of flowering remained stable over time. A decrease in mean seed production per fruit with increasing flowering time suggests selection against late bloomers. However, germination rate was not affected by seed collection date, and the main seed disperser, the bat Eidolon helvum, seemed to increase in abundance at the end of the reproductive season and preferred trees in open habitats where early and late bloomers are expected. CONCLUSIONS The pairwise approach performs well in detecting IBT. The persistence of different mating pools in such a case may result from a trade off between selective forces during the mating and seed dispersal processes.

Soil Charcoal to Assess the Impacts of Past Human Disturbances on Tropical Forests

The canopy of many central African forests is dominated by light-demanding tree species that do not regenerate well under themselves. The prevalence of these species might result from ancient slash-and-burn agricultural activities that created large openings, while a decline of these activities since the colonial period could explain their deficit of regeneration. To verify this hypothesis, we compared soil charcoal abundance, used as a proxy for past slash-and-burn agriculture, and tree species composition assessed on 208 rainforest 0.2 ha plots located in three areas from Southern Cameroon. Species were classified in regeneration guilds (pioneer, non-pioneer light-demanding, shade-bearer) and characterized by their wood-specific gravity, assumed to reflect light requirement. We tested the correlation between soil charcoal abundance and: (i) the relative abundance of each guild, (ii) each species and family abundance and (iii) mean wood-specific gravity. Charcoal was found in 83% of the plots, indicating frequent past forest fires. Radiocarbon dating revealed two periods of fires: “recent” charcoal were on average 300 years old (up to 860 BP, n = 16) and occurred in the uppermost 20 cm soil layer, while “ancient” charcoal were on average 1900 years old (range: 1500 to 2800 BP, n = 43, excluding one sample dated 9400 BP), and found in all soil layers. While we expected a positive correlation between the relative abundance of light-demanding species and charcoal abundance in the upper soil layer, overall there was no evidence that the current heterogeneity in tree species composition can be explained by charcoal abundance in any soil layer. The absence of signal supporting our hypothesis might result from (i) a relatively uniform impact of past slash-and-burn activities, (ii) pedoturbation processes bringing ancient charcoal to the upper soil layer, blurring the signal of centuries-old Human disturbances, or (iii) the prevalence of other environmental factors on species composition.

Speciation slowing down in widespread and long-living tree taxa: insights from the tropical timber tree genus Milicia (Moraceae)

The long generation time and large effective size of widespread forest tree species can result in slow evolutionary rate and incomplete lineage sorting, complicating species delimitation. We addressed this issue with the African timber tree genus Milicia that comprises two morphologically similar and often confounded species: M. excelsa, widespread from West to East Africa, and M. regia, endemic to West Africa. We combined information from nuclear microsatellites (nSSRs), nuclear and plastid DNA sequences, and morphological systematics to identify significant evolutionary units and infer their evolutionary and biogeographical history. We detected five geographically coherent genetic clusters using nSSRs and three levels of genetic differentiation. First, one West African cluster matched perfectly with the morphospecies M. regia that formed a monophyletic clade at both DNA sequences. Second, a West African M. excelsa cluster formed a monophyletic group at plastid DNA and was more related to M. regia than to Central African M. excelsa, but shared many haplotypes with the latter at nuclear DNA. Third, three Central African clusters appeared little differentiated and shared most of their haplotypes. Although gene tree paraphyly could suggest a single species in Milicia following the phylogenetic species concept, the existence of mutual haplotypic exclusivity and nonadmixed genetic clusters in the contact area of the two taxa indicate strong reproductive isolation and, thus, two species following the biological species concept. Molecular dating of the first divergence events showed that speciation in Milicia is ancient (Tertiary), indicating that long-living tree taxa exhibiting genetic speciation may remain similar morphologically.

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

BackgroundSpecies 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.ResultsWest 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.ConclusionsOur 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.

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.

High spatial resolution of late-Holocene human activities in the moist forests of Central Africa using soil charcoal and charred botanical remains

Palaeoecological and archaeological studies have demonstrated that human populations have long inhabited the moist forests of central Africa. However, spatial and temporal patterns of human activities have hardly been investigated with satisfactory accuracy. In this study, we propose to characterize past human activities at local scale by using a systematic quantitative and qualitative methodology based on soil charcoal and charred botanical remains. A total of 88 equidistant test-pits were excavated along six transects in two contrasting forest types in southern Cameroon. Charred botanical remains were collected by water-sieving and sorted by type (wood charcoals, oil palm endocarps and unidentified seeds). A total of 50 Accelerator Mass Spectrometry 14C dates were also obtained. Results showed that charred macroremains were found at multiple places in the forest, suggesting scattered human activities, which were distributed into two main periods (Phase A: 2300–1300 BP; Phase B: 580 BP to the present). Charred botanical remains indicated two types of land-use: (1) domestic, with oil palm endocarps most often associated with potsherds (villages) and (2) agricultural, with charcoal as probable remnant of slash-and-burn cultivation (fields). Oil palm endocarp abundance decreased with distance from the identified human settlements. Our methodology allowed documenting, at high resolution, the spatial and temporal patterns of human activities in central African moist forests and could be applied to other tropical contexts.