The African baobab (Adansonia digitata, Malvaceae): genetic resources in neglected populations of the Nuba Mountains, Sudan.

UNLABELLED: • PREMISE OF THE STUDY: Adansonia digitata L. is one of the most important indigenous fruit trees of mainland Africa. Despite its significance for subsistence and income generation of local communities, little is known about the genetic and morphological variability of East African populations of A. digitata, including those of Sudan. The aim of the current study, therefore, was to analyze genetic and morphological variability of different baobab populations in Kordofan, Sudan and to estimate the effect of human intervention on genetic differentiation and diversity.• METHODS: A total of 306 trees were randomly sampled from seven spatially separated locations in the Nuba Mountains, Sudan, to cover a wide range of differing environmental gradients and management regimes ('homesteads' and 'wild'). Genetic analyses were conducted using nine microsatellite markers. Because of the tetraploid nature of A. digitata, different approaches were applied to estimate patterns of genetic diversity. Investigations were completed by measurements of dendrometric and fruit morphological characters.• KEY RESULTS: Genetic diversity was balanced and did not differ between locations or management regimes, although tendencies of higher diversity in 'homesteads' were observed. A Bayesian cluster approach detected two distinct gene pools in the sample set, mainly caused by one highly diverse population close to a main road. The variability of tree characters and fruit morphometries was high, and significantly different between locations.• CONCLUSIONS: Results indicated a rather positive effect with human intervention. The observed populations provide a promising gene pool and likely comprise ecotypes well-adapted to environmental conditions at the northern distribution range of the species, which should be considered in conservation and management programs.

Nuclear and chloroplast DNA phylogeography reveals Pleistocene divergence and subsequent secondary contact of two genetic lineages of the tropical rainforest tree species Shorea leprosula (Dipterocarpaceae) in South-East Asia.

Tropical rainforests in South-East Asia have been affected by climatic fluctuations during past glacial eras. To examine how the accompanying changes in land areas and temperature have affected the genetic properties of rainforest trees in the region, we investigated the phylogeographic patterns of a widespread dipterocarp species, Shorea leprosula. Two types of DNA markers were used: expressed sequence tag-based simple sequence repeats and chloroplast DNA (cpDNA) sequence variations. Both sets of markers revealed clear genetic differentiation between populations in Borneo and those in the Malay Peninsula and Sumatra (Malay/Sumatra). However, in the south-western part of Borneo, genetic admixture of the lineages was observed in the two marker types. Coalescent simulation based on cpDNA sequence variation suggested that the two lineages arose 0.28-0.09 million years before present and that following their divergence migration from Malay/Sumatra to Borneo strongly exceeded migration in the opposite direction. We conclude that the genetic structure of S. leprosula was largely formed during the middle Pleistocene and was subsequently modified by eastward migration across the subaerially exposed Sunda Shelf.

Development of microsatellite markers for Anadenanthera colubrina var. cebil (Fabaceae), a native tree from South America.

PREMISE OF THE STUDY: Microsatellite primers were developed in the native legume tree Anadenanthera colubrina var. cebil to study the genetic diversity and genetic structure in natural populations in Argentina. METHODS AND RESULTS: Nine microsatellite markers were identified using a genomic library enriched for tandemly repeated motifs, eight of which markers were polymorphic. The polymorphism of these markers was assessed by investigating 20 individuals for fragment polymorphism; three to 13 alleles were observed for each locus. Observed and expected heterozygosities ranged from 0.300 to 1.000 and from 0.463 to 0.900, respectively. CONCLUSIONS: These results confirm that these primers will be useful for investigating the genetic diversity and genetic structure of natural populations of A. colubrina var. cebil in future studies.

Host plant richness and environment in tropical forest transformation systems shape arbuscular mycorrhizal fungal richness.

Transformation of tropical lowland rain forests into rubber tree and oil palm plantations is the cause of massive loss of vegetation diversity. The consequences for associated mycorrhizal fungi are not fully understood. We hypothesized that generalist arbuscular mycorrhizal fungi are resistant to removal of host species richness and that forest conversion to oil palm and rubber leads to loss of arbuscular mycorrhizal fungal (AMF) species with host preferences. Plant identities and AMF species were determined by molecular barcoding of 112 roots collected in three land-use systems (rain forest, rubber tree and oil palm plantation) in two landscapes on Sumatra (Indonesia), a world hotspot of forest transformation. The collected roots were from 43 forest plant species, in addition to rubber trees and oil palms. We detected 28 AMF species of which about 75% were present in forest trees and 25% shared among the land use systems. Only one AMF species present in plantation roots was not detected in the analyzed forest roots. Host specificity of arbuscular mycorrhizal fungi was not detected. Oil palm and rubber tree roots exhibited a strong reduction in AMF richness compared with roots from rainforests and were differentiated by soil resources. On basis of an individual root, oil palm had a lower AMF species richness than forest or rubber tree roots. Our results demonstrate that tropical AMF communities are shaped by two mechanisms: (i) root habitat diversity as the result of plant diversity and (ii) habitat properties as the result of plant traits or environmental conditions and management. Collectively, deterioration of habitat diversity and properties exacerbates impoverishment of AMF assemblages.

Mutation rates in scots pine (Pinus sylvestris L.) from the Chernobyl exclusion zone evaluated with amplified fragment-length polymorphisms (AFLPs) and microsatellite markers.

Ionizing radiation is a strong mutagenic factor and, accordingly, elevated mutation rates would be expected in plants exposed to high chronic or acute radiation after the Chernobyl accident in 1986. Somatic mutations were analyzed in pines (Pinus sylvestris L.) planted before and after the Chernobyl accident and in control material of the same origin planted in sites with natural radiation. Microsatellites (SSRs) and amplified fragment-length polymorphisms (AFLPs) were investigated. The mutation rates for microsatellites were estimated as 2.8 × 10(-4)-7.1 × 10(-4) per locus for different irradiated tree populations; no mutations were detected in the controls. In the case of AFLPs, the observed mutation rates were 3.74 × 10(-3) -3.99 × 10(-3) and 1.06 × 10(-3) per locus for contaminated and control areas, respectively. Thus a statistically highly significant three-fold increase in number of mutations was found by the use of AFLP markers, indicating that ionizing radiation causes strong DNA damage across the entire genome and that AFLPs may be the appropriate marker system for this kind of analysis.

Molecular database for classifying Shorea species (Dipterocarpaceae) and techniques for checking the legitimacy of timber and wood products.

The extent of tropical forest has been declining, due to over-exploitation and illegal logging activities. Large quantities of unlawfully extracted timber and other wood products have been exported, mainly to developed countries. As part of the export monitoring effort, we have developed methods for extracting and analyzing DNA from wood products, such as veneers and sawn timbers made from dipterocarps, in order to identify the species from which they originated. We have also developed a chloroplast DNA database for classifying Shorea species, which are both ecologically and commercially important canopy tree species in the forests of Southeast Asia. We are able to determine the candidate species of wood samples, based on DNA sequences and anatomical data. The methods for analyzing DNA from dipterocarp wood products may have strong deterrent effects on international trade of illegitimate dipterocarp products. However, the method for analyzing DNA from wood is not perfect for all wood products and need for more improvement, especially for plywood sample. Consequently, there may be benefits for the conservation of tropical forests in Southeast Asia.

A candidate gene association analysis identifies SNPs potentially involved in drought tolerance in European beech (Fagus sylvatica L.).

Studies of genetic variation underlying traits related to drought tolerance in forest trees are of great importance for understanding their adaptive potential under a climate change scenario. In this study, using a candidate gene approach, associations between SNPs and drought related traits were assessed in saplings of European beech (Fagus sylvatica L.) representing trees growing along steep precipitation gradients. The saplings were subjected to experimentally controlled drought treatments. Response of the saplings was assessed by the evaluation of stem diameter growth (SDG) and the chlorophyll fluorescence parameters FV/FM, PIabs, and PItot. The evaluation showed that saplings from xeric sites were less affected by the drought treatment. Five SNPs (7.14%) in three candidate genes were significantly associated with the evaluated traits; saplings with particular genotypes at these SNPs showed better performance under the drought treatment. The SNPs were located in the cytosolic class I small heat-shock protein, CTR/DRE binding transcription factor, and isocitrate dehydrogenase genes and explained 5.8-13.4% of the phenotypic variance. These findings provide insight into the genetic basis of traits related to drought tolerance in European beech and could support the development of forest conservation management strategies under future climatic conditions.

Molecular genetic tools to infer the origin of forest plants and wood.

Most forest tree species exhibit high levels of genetic diversity that can be used to trace the origin of living plants or their products such as timber and processed wood. Recent progress to isolate DNA not only from living tissue but also from wood and wood products offers new opportunities to test the declared origin of material such as seedlings for plantation establishment or timber. However, since most forest tree populations are weakly differentiated, the identification of genetic markers to differentiate among spatially isolated populations is often difficult and time consuming. Two important fields of "forensic" applications are described: Molecular tools are applied to test the declared origin of forest reproductive material used for plantation establishment and of internationally traded timber and wood products. These applications are illustrated taking examples from Germany, where mechanisms have been developed to improve the control of the trade with forest seeds and seedlings, and from the trade with wood of the important Southeast Asian tree family Dipterocarpaceae. Prospects and limitations of the use of molecular genetic methods to conclude on the origin of forest plants, wood, and wood products are discussed.

Patterns of contemporary hybridization inferred from paternity analysis in a four-oak-species forest.

BACKGROUND: Few studies address the issue of hybridization in a more than two-species context. The species-rich Quercus complex is one of the systems which can offer such an opportunity. To investigate the contemporary pattern of hybridization we sampled and genotyped 320 offspring from a natural mixed forest comprising four species of the European white oak complex: Quercus robur, Q. petraea, Q. pubescens, and Q. frainetto. RESULTS: A total of 165 offspring were assigned unambiguously to one of the pollen donors within the study plot. The minimum amount of effective pollen originating from outside the plot varied markedly among the seed parents, ranging from 0.18 to 0.87. The majority of the successful matings (64.1%) occurred between conspecific individuals indicating the existence of reproductive barriers between oak species. However, the isolation was not complete since we found strong evidence for both first-generation (8.4%) and later-generation hybrids (27.5%). Only two out of eight seed parents, belonging to Q. petraea and Q. robur, showed a high propensity to hybridize with Q. pubescens and Q. petraea, respectively. Significant structure of the effective pollen pools (Phipt=0.069, P=0.01) was detected in our sample. However, no support was found for the isolation by distance hypothesis. The proportion of hybrids was much higher (79%) in the seed generation when compared to the adult tree generation. CONCLUSION: First-generation hybrids were observed only between three out of six possible species combinations. Hybrids between one pair of species preferred to mate with one of their parental species. The observation of first and later-generation hybrids in higher frequency in acorns than in adults might be explained by selection against hybrid genotypes, the history of this uneven-aged forest or past introgression between species.

Genetic and genomic approaches to assess adaptive genetic variation in plants: forest trees as a model.

With the increasing availability of sequence information at putatively important genes or regulatory regions, the characterization of adaptive genetic diversity and their association with phenotypic trait variation becomes feasible for many non-model organisms such as forest trees. Especially in predominantly outcrossing forest tree populations with large effective size, a high genetic variation in relevant genes is maintained, that is the raw material for the adaptation to changing and variable environments, and likewise for plant breeding. Oaks (Quercus spp.) are excellent model species to study the adaptation of forest trees to changing environments. They show a wide geographic distribution in Europe as dominant tree species in many forests and grow under a wide range of climatic and edaphic conditions. With the availability of a growing amount of functional and expressional candidate genes, we are now able to test the functional importance of single nucleotide polymorphisms (SNPs) by associating nucleotide variation in these genes with phenotypic variation in adaptive traits in segregating or natural populations. Here, we report on quantitative trait locus (QTL), candidate gene and association mapping approaches that are applicable to characterize gene markers and SNPs associated with variation in adaptive traits, such as bud burst, drought resistance and other traits showing selective responses to environmental change and stress. Because genome-wide association mapping studies are not feasible because of the enormous amount of SNP markers required in outcrossing trees with high recombination rates, the success of such an approach depends largely on the reasonable selection of candidate genes.

Transferability of Simple Sequence Repeat (SSR) Markers Developed in Litchi chinensis to Blighia sapida (Sapindaceae).

Ackee (Blighia sapida, Sapindaceae) is a multipurpose fruit tree species of high economic importance, native to the Guinean forests of West Africa, and belongs to the same family as that of lychee (Litchi chinensis). In this study, a set of 12 primer pairs for simple sequence repeats (SSRs) previously developed for lychee has been evaluated for polymorphism in 16 ackee trees from different populations. Seven primer pairs have been found to be transferable, and four have revealed polymorphisms. However, the average number of alleles per locus has dropped from 4.9 for lychee to 3.7 for ackee. Characterization of the four polymorphic markers in 279 individuals belonging to14 different ackee populations from Benin has revealed that the numbers of alleles per locus range from two to 14 with a mean number of 5.8. The observed and expected heterozygosities range between 0.020 to 0.359 and 0.020 to 0.396, respectively.

DNA barcoding of flowering plants in Sumatra, Indonesia.

The rapid conversion of Southeast Asian lowland rainforests into monocultures calls for the development of rapid methods for species identification to support ecological research and sustainable land-use management. Here, we investigated the utilization of DNA barcodes for identifying flowering plants from Sumatra, Indonesia. A total of 1,207 matK barcodes (441 species) and 2,376 rbcL barcodes (750 species) were successfully generated. The barcode effectiveness is assessed using four approaches: (a) comparison between morphological and molecular identification results, (b) best-close match analysis with TaxonDNA, (c) barcoding gap analysis, and (d) formation of monophyletic groups. Results show that rbcL has a much higher level of sequence recoverability than matK (95% and 66%). The comparison between morphological and molecular identifications revealed that matK and rbcL worked best assigning a plant specimen to the genus level. Estimates of identification success using best-close match analysis showed that >70% of the investigated species were correctly identified when using single barcode. The use of two-loci barcodes was able to increase the identification success up to 80%. The barcoding gap analysis revealed that neither matK nor rbcL succeeded to create a clear gap between the intraspecific and interspecific divergences. However, these two barcodes were able to discriminate at least 70% of the species from each other. Fifteen genera and twenty-one species were found to be nonmonophyletic with both markers. The two-loci barcodes were sufficient to reconstruct evolutionary relationships among the plant taxa in the study area that are congruent with the broadly accepted APG III phylogeny.

Evidence for hybridization and introgression within a species-rich oak (Quercus spp.) community.

BACKGROUND: Analysis of interspecific gene flow is crucial for the understanding of speciation processes and maintenance of species integrity. Oaks (genus Quercus, Fagaceae) are among the model species for the study of hybridization. Natural co-occurrence of four closely related oak species is a very rare case in the temperate forests of Europe. We used both morphological characters and genetic markers to characterize hybridization in a natural community situated in west-central Romania and which consists of Quercus robur, Q. petraea, Q. pubescens, and Q. frainetto, respectively. RESULTS: On the basis of pubescence and leaf morphological characters ~94% of the sampled individuals were assigned to pure species. Only 16 (approximately 6%) individual trees exhibited intermediate morphologies or a combination of characters of different species. Four chloroplast DNA haplotypes were identified in the study area. The distribution of haplotypes within the white oak complex showed substantial differences among species. However, the most common haplotypes were present in all four species. Furthermore, based on a set of 7 isozyme and 6 microsatellite markers and using a Bayesian admixture analysis without any a priori information on morphology we found that four genetic clusters best fit the data. There was a very good correspondence of each species with one of the inferred genetic clusters. The estimated introgression level varied markedly between pairs of species ranging from 1.7% between Q. robur and Q. frainetto to 16.2% between Q. pubescens and Q. frainetto. Only nine individuals (3.4%) appeared to be first-generation hybrids. CONCLUSION: Our data indicate that natural hybridization has occurred at relatively low rates. The different levels of gene flow among species might be explained by differences in flowering time and spatial position within the stand. In addition, a partial congruence between phenotypically and genetically intermediate individuals was found, suggesting that intermediate appearance does not necessarily mean hybridization. However, it appears that natural hybridization did not seriously affect the species identity in this area of sympatry.

Modeling population genetic data in autotetraploid species.

Allozyme and PCR-based molecular markers have been widely used to investigate genetic diversity and population genetic structure in autotetraploid species. However, an empirical but inaccurate approach was often used to infer marker genotype from the pattern and intensity of gel bands. Obviously, this introduces serious errors in prediction of the marker genotypes and severely biases the data analysis. This article developed a theoretical model to characterize genetic segregation of alleles at genetic marker loci in autotetraploid populations and a novel likelihood-based method to estimate the model parameters. The model properly accounts for segregation complexities due to multiple alleles and double reduction at autotetrasomic loci in natural populations, and the method takes appropriate account of incomplete marker phenotype information with respect to genotype due to multiple-dosage allele segregation at marker loci in tetraploids. The theoretical analyses were validated by making use of a computer simulation study and their utility is demonstrated by analyzing microsatellite marker data collected from two populations of sycamore maple (Acer pseudoplatanus L.), an economically important autotetraploid tree species. Numerical analyses based on simulation data indicate that the model parameters can be adequately estimated and double reduction is detected with good power using reasonable sample size.

Gene flow of common ash (Fraxinus excelsior L.) in a fragmented landscape.

Gene flow dynamics of common ash (Fraxinus excelsior L.) is affected by several human activities in Central Europe, including habitat fragmentation, agroforestry expansion, controlled and uncontrolled transfer of reproductive material, and a recently introduced emerging infectious disease, ash dieback, caused by Hymenoscyphus fraxineus. Habitat fragmentation may alter genetic connectivity and effective population size, leading to loss of genetic diversity and increased inbreeding in ash populations. Gene flow from cultivated trees in landscapes close to their native counterparts may also influence the adaptability of future generations. The devastating effects of ash dieback have already been observed in both natural and managed populations in continental Europe. However, potential long-term effects of genetic bottlenecks depend on gene flow across fragmented landscapes. For this reason, we studied the genetic connectivity of ash trees in an isolated forest patch of a fragmented landscape in Rösenbeck, Germany. We applied two approaches to parentage analysis to estimate gene flow patterns at the study site. We specifically investigated the presence of background pollination at the landscape level and the degree of genetic isolation between native and cultivated trees. Local meteorological data was utilized to understand the effect of wind on the pollen and seed dispersal patterns. Gender information of the adult trees was considered for calculating the dispersal distances. We found that the majority of the studied seeds (55-64%) and seedlings (75-98%) in the forest patch were fathered and mothered by the trees within the same patch. However, we determined a considerable amount of pollen flow (26-45%) from outside of the study site, representing background pollination at the landscape level. Limited pollen flow was observed from neighbouring cultivated trees (2%). Both pollen and seeds were dispersed in all directions in accordance with the local wind directions. Whereas there was no positive correlation between pollen dispersal distance and wind speed, the correlation between seed dispersal distance and wind speed was significant (0.71, p < 0.001), indicating that strong wind favours long-distance dispersal of ash seeds. Finally, we discussed the implications of establishing gene conservation stands and the use of enrichment planting in the face of ash dieback.

De novo transcriptome assembly and analysis of differential gene expression in response to drought in European beech.

Despite the ecological and economic importance of European beech (Fagus sylvatica L.) genomic resources of this species are still limited. This hampers an understanding of the molecular basis of adaptation to stress. Since beech will most likely be threatened by the consequences of climate change, an understanding of adaptive processes to climate change-related drought stress is of major importance. Here, we used RNA-seq to provide the first drought stress-related transcriptome of beech. In a drought stress trial with beech saplings, 50 samples were taken for RNA extraction at five points in time during a soil desiccation experiment. De novo transcriptome assembly and analysis of differential gene expression revealed 44,335 contigs, and 662 differentially expressed genes between the stress and normally watered control group. Gene expression was specific to the different time points, and only five genes were significantly differentially expressed between the stress and control group on all five sampling days. GO term enrichment showed that mostly genes involved in lipid- and homeostasis-related processes were upregulated, whereas genes involved in oxidative stress response were downregulated in the stressed seedlings. This study gives first insights into the genomic drought stress response of European beech, and provides new genetic resources for adaptation research in this species.

Population Genetic Structure and Reproductive Strategy of the Introduced Grass Centotheca lappacea in Tropical Land-Use Systems in Sumatra.

Intensive transformation of lowland rainforest into oil palm and rubber monocultures is the most common land-use practice in Sumatra (Indonesia), accompanied by invasion of weeds. In the Jambi province, Centotheca lappacea is one of the most abundant alien grass species in plantations and in jungle rubber (an extensively used agroforest), but largely missing in natural rainforests. Here, we investigated putative genetic differentiation and signatures for adaptation in the introduced area. We studied reproductive mode and ploidy level as putative factors for invasiveness of the species. We sampled 19 populations in oil palm and rubber monocultures and in jungle rubber in two regions (Bukit Duabelas and Harapan). Amplified fragment length polymorphisms (AFLP) revealed a high diversity of individual genotypes and only a weak differentiation among populations (FST = 0.173) and between the two regions (FST = 0.065). There was no significant genetic differentiation between the three land-use systems. The metapopulation of C. lappacea consists of five genetic partitions with high levels of admixture; all partitions appeared in both regions, but with different proportions. Within the Bukit Duabelas region we observed significant isolation-by-distance. Nine AFLP loci (5.3% of all loci) were under natural diversifying selection. All studied populations of C. lappacea were diploid, outcrossing and self-incompatible, without any hints of apomixis. The estimated residence time of c. 100 years coincides with the onset of rubber and oil palm planting in Sumatra. In the colonization process, the species is already in a phase of establishment, which may be enhanced by efficient selection acting on a highly diverse gene pool. In the land-use systems, seed dispersal might be enhanced by adhesive spikelets. At present, the abundance of established populations in intensively managed land-use systems might provide opportunities for rapid dispersal of C. lappacea across rural landscapes in Sumatra, while the invasion potential in rainforest ecosystems appears to be moderate as long as they remain undisturbed.

Land-use choices follow profitability at the expense of ecological functions in Indonesian smallholder landscapes.

Smallholder-dominated agricultural mosaic landscapes are highlighted as model production systems that deliver both economic and ecological goods in tropical agricultural landscapes, but trade-offs underlying current land-use dynamics are poorly known. Here, using the most comprehensive quantification of land-use change and associated bundles of ecosystem functions, services and economic benefits to date, we show that Indonesian smallholders predominantly choose farm portfolios with high economic productivity but low ecological value. The more profitable oil palm and rubber monocultures replace forests and agroforests critical for maintaining above- and below-ground ecological functions and the diversity of most taxa. Between the monocultures, the higher economic performance of oil palm over rubber comes with the reliance on fertilizer inputs and with increased nutrient leaching losses. Strategies to achieve an ecological-economic balance and a sustainable management of tropical smallholder landscapes must be prioritized to avoid further environmental degradation.

Characterization and transferability of microsatellite markers developed for Carpinus betulus (Betulaceae)(1).

PREMISE OF THE STUDY: Carpinus betulus (Betulaceae) is an octoploid, ecologically important, common tree species in European woodlands. We established 11 nuclear microsatellite loci allowing for detailed analyses of genetic diversity and structure. METHODS AND RESULTS: A microsatellite-enriched library was used to develop primers for 11 microsatellite loci that revealed high allele numbers and genetic diversity in a preliminary study. CONCLUSIONS: All of the loci developed here are informative for C. betulus. In addition, the loci are transferable to several species within the genus, and almost all loci cross-amplified in species of different genera of the Betulaceae.

Genetic diversity in aspen and its relation to arthropod abundance.

The ecological consequences of biodiversity have become a prominent public issue. Little is known on the effect of genetic diversity on ecosystem services. Here, a diversity experiment was established with European and North American aspen (Populus tremula, P. tremuloides) planted in plots representing either a single deme only or combinations of two, four and eight demes. The goals of this study were to explore the complex inter- and intraspecific genetic diversity of aspen and to then relate three measures for diversity (deme diversity, genetic diversity determined as Shannon index or as expected heterozygosity) to arthropod abundance. Microsatellite and AFLP markers were used to analyze the genetic variation patterns within and between the aspen demes and deme mixtures. Large differences were observed regarding the genetic diversity within demes. An analysis of molecular variance revealed that most of the total genetic diversity was found within demes, but the genetic differentiation among demes was also high. The complex patterns of genetic diversity and differentiation resulted in large differences of the genetic variation within plots. The average diversity increased from plots with only one deme to plots with two, four, and eight demes, respectively and separated plots with and without American aspen. To test whether intra- and interspecific diversity impacts on ecosystem services, arthropod abundance was determined. Increasing genetic diversity of aspen was related to increasing abundance of arthropods. However, the relationship was mainly driven by the presence of American aspen suggesting that species identity overrode the effect of intraspecific variation of European aspen.