Bernd Degen

Fine‐scale genetic structure and gene dispersal inferences in 10 Neotropical tree species

The extent of gene dispersal is a fundamental factor of the population and evolutionary dynamics of tropical tree species, but directly monitoring seed and pollen movement is a difficult task. However, indirect estimates of historical gene dispersal can be obtained from the fine‐scale spatial genetic structure of populations at drift–dispersal equilibrium. Using an approach that is based on the slope of the regression of pairwise kinship coefficients on spatial distance and estimates of the effective population density, we compare indirect gene dispersal estimates of sympatric populations of 10 tropical tree species. We re‐analysed 26 data sets consisting of mapped allozyme, SSR (simple sequence repeat), RAPD (random amplified polymorphic DNA) or AFLP (amplified fragment length polymorphism) genotypes from two rainforest sites in French Guiana. Gene dispersal estimates were obtained for at least one marker in each species, although the estimation procedure failed under insufficient marker polymorphism, limited sample size, or inappropriate sampling area. Estimates generally suffered low precision and were affected by assumptions regarding the effective population density. Averaging estimates over data sets, the extent of gene dispersal ranged from 150 m to 1200 m according to species. Smaller gene dispersal estimates were obtained in species with heavy diaspores, which are presumably not well dispersed, and in populations with high local adult density. We suggest that limited seed dispersal could indirectly limit effective pollen dispersal by creating higher local tree densities, thereby increasing the positive correlation between pollen and seed dispersal distances. We discuss the potential and limitations of our indirect estimation procedure and suggest guidelines for future studies.

High level of variation at Abies alba chloroplast microsatellite loci in Europe

Based on two polymorphic chloroplast microsatellites that had been previously identified and sequence characterized in the genus Abies, genetic variation was studied in a total of 714 individuals from 17 European silver fir (Abies alba Mill.) populations distributed all over the natural range. We found eight and 18 different length variants at each locus, respectively, which combined into 90 different haplotypes. Genetic distances between most populations were high and significant. There is also evidence for spatial organization of the distribution of haplotypes, as shown by permutation tests, which demonstrate that genetic distances increase with spatial distances. A large heterogeneity in levels of diversity across populations was observed. Furthermore, there is good congruence in the levels of allelic richness of the two loci across populations. The present organization of levels of allelic richness across the range of the species is likely to have been shaped by the distribution of refugia during the last glaciation and the subsequent recolonization processes.

Optimal sampling strategy for estimation of spatial genetic structure in tree populations

Fine-scale spatial genetic structure (SGS) in natural tree populations is largely a result of restricted pollen and seed dispersal. Understanding the link between limitations to dispersal in gene vectors and SGS is of key interest to biologists and the availability of highly variable molecular markers has facilitated fine-scale analysis of populations. However, estimation of SGS may depend strongly on the type of genetic marker and sampling strategy (of both loci and individuals). To explore sampling limits, we created a model population with simulated distributions of dominant and codominant alleles, resulting from natural regeneration with restricted gene flow. SGS estimates from subsamples (simulating collection and analysis with amplified fragment length polymorphism (AFLP) and microsatellite markers) were correlated with the ‘real’ estimate (from the full model population). For both marker types, sampling ranges were evident, with lower limits below which estimation was poorly correlated and upper limits above which sampling became inefficient. Lower limits (correlation of 0.9) were 100 individuals, 10 loci for microsatellites and 150 individuals, 100 loci for AFLPs. Upper limits were 200 individuals, five loci for microsatellites and 200 individuals, 100 loci for AFLPs. The limits indicated by simulation were compared with data sets from real species. Instances where sampling effort had been either insufficient or inefficient were identified. The model results should form practical boundaries for studies aiming to detect SGS. However, greater sample sizes will be required in cases where SGS is weaker than for our simulated population, for example, in species with effective pollen/seed dispersal mechanisms.

Limited pollen dispersal and biparental inbreeding in Symphonia globulifera in French Guiana

In this paper, we report a study of the mating system and gene flow of Symphonia globulifera, a hermaphroditic, mainly bird-pollinated tree species with a large geographic distribution in the tropical Americas and Africa. Using three microsatellites, we analysed 534 seeds of 28 open pollinated families and 164 adults at the experimental site ‘Paracou’ in French Guiana. We observed, compared to other tropical tree species, relatively high values for the effective number of alleles. Significant spatial genetic structure was detected, with trees at distances up to 150 m more genetically similar than expected at random. We estimated parameters of the mating system and gene flow by using the mixed mating model and the TwoGener approach. The estimated multilocus outcrossing rate, tm, was 0.920. A significant level of biparental inbreeding and a high proportion of full-sibs were estimated for the 28 seed arrays. We estimated mean pollen dispersal distances between 27 and 53 m according to the dispersal models used. Although the adult population density of S. globulifera in Paracou was relatively high, the joint estimation of pollen dispersal and density of reproductive trees gave effective density estimates of 1.6 and 1.3 trees/ha. The parameters of the mating system and gene flow are discussed in the context of spatial genetic and demographic structures, flowering phenology and pollinator composition and behaviour.

Fine-scale spatial genetic structure of eight tropical tree species as analysed by RAPDs

The fine-scale spatial genetic structure of eight tropical tree species (Chrysophyllum sanguinolentum, Carapa procera, Dicorynia guianensis, Eperua grandiflora, Moronobea coccinea, Symphonia globulifera, Virola michelii, Vouacapoua americana) was studied in populations that were part of a silvicultural trial in French Guiana. The species analysed have different spatial distribution, sexual system, pollen and seed dispersal agents, flowering phenology and environmental demands. The spatial position of trees and a RAPD data set for each species were combined using a multivariate genetic distance method to estimate spatial genetic structure. A significant spatial genetic structure was found for four of the eight species. In contrast to most observations in temperate forests, where spatial structure is not usually detected at distances greater than 50 m, significant genetic structure was found at distances up to 300 m. The relationships between spatial genetic structure and life history characteristics are discussed.

Comparative study of genetic variation and differentiation of two pedunculate oak (Quercus robur) stands using microsatellite and allozyme loci

In a comparative study four codominant microsatellite loci and seven allozyme gene loci have been used to investigate the genetic variation and differentiation of two pedunculate oak stands in North Germany. Both number and effective number of alleles were five to six times higher and the observed heterozygosity was three times higher for the microsatellite than for the allozyme loci. One stand showed an overall excess of homozygotes. In general the microsatellites were closer to Hardy–Weinberg expectation. The genetic distances between the two stands were distinctly higher for microsatellites. For most parameters microsatellites exhibited smaller interlocus variation than the allozymes. The different impact of population genetic processes on the genetic structure as assessed by microsatellites or allozymes is discussed.

Chloroplast DNA variation of oaks in western Central Europe and genetic consequences of human influences

Oak chloroplast DNA (cpDNA) variation was studied in a grid-based inventory in western Central Europe, including Belgium, The Netherlands, Luxembourg, Germany, the Czech Republic, and the northern parts of Upper and Lower Austria. A total of 2155 trees representing 426 populations of Quercus robur L. and Q. petraea (Matt.) Liebl. were screened for polymorphism in up to four PCR-amplified cpDNA fragments. Eleven haplotypes belonging to four lineages were detected; these lineages were formerly restricted to glacial refugia in the Iberian Peninsula, the Apennine Peninsula and the Balkan Peninsula. The haplotypes originating from the Apennines are particularly well represented in the study region, but there is also a significant contribution from the other refugia, which explain the high overall level of cpDNA diversity. The strong human impact in western Central Europe during the past centuries, which has resulted in the clearance of most forests, was followed by reforestation, sometimes involving seed transfers. Despite this strong human impact, broad geographic patterns of lineages and haplotypes could still be detected. To evaluate further the consequences of the former human activities on the present day oak cpDNA genetic structure, four regions where increasingly strong human impact was anticipated (ranging from hilly regions in southern Germany to roadsides plantations in The Netherlands) were selected. There, a comparison of the levels of intrapopulation cpDNA diversity and spatial structuring was made. Over the whole area, within stand diversity was significantly higher in Q. robur than in Q. petraea (hS ¼ 0:24 vs. 0.16). Since total diversity is identical for both species, this results in a significantly lower level of fixation for Q. robur than for Q. petraea (GST ¼ 0:68 vs. 0.79). The analyses also reveal a decrease of fixation with increasing human impact on oak populations. The Dutch roadside plantations (Q. robur) exhibit a very low level of fixation ðGST ¼ 0:28Þ as compared to Q. petraea in southern Germany ðGST ¼ 0:91Þ. The significance of the spatial genetic structure was tested using geostatistical methods. For the complete data set, a strong spatial genetic structure is confirmed, with higher than average genetic similarities between populations distant from up to 270 km, whereas there is no spatial structure in the roadside plantations in The Netherlands or in the northern German lowlands. These results should help to differentiate introduced from autochthonous populations, and provide a framework for the identification of the geographic origin of seed lots. # 2002 Elsevier Science B.V. All rights reserved.

Verifying the geographic origin of mahogany (Swietenia macrophylla King) with DNA-fingerprints

Illegal logging is one of the main causes of ongoing worldwide deforestation and needs to be eradicated. The trade in illegal timber and wood products creates market disadvantages for products from sustainable forestry. Although various measures have been established to counter illegal logging and the subsequent trade, there is a lack of practical mechanisms for identifying the origin of timber and wood products. In this study, six nuclear microsatellites were used to generate DNA fingerprints for a genetic reference database characterising the populations of origin of a large set of mahogany (Swietenia macrophylla King, Meliaceae) samples. For the database, leaves and/or cambium from 1971 mahogany trees sampled in 31 stands from Mexico to Bolivia were genotyped. A total of 145 different alleles were found, showing strong genetic differentiation (δ(Gregorious)=0.52, F(ST)=0.18, G(ST(Hedrick))=0.65) and clear correlation between genetic and spatial distances among stands (r=0.82, P<0.05). We used the genetic reference database and Bayesian assignment testing to determine the geographic origins of two sets of mahogany wood samples, based on their multilocus genotypes. In both cases the wood samples were assigned to the correct country of origin. We discuss the overall applicability of this methodology to tropical timber trading.

Use of DNA fingerprints to control the origin of sapelli timber (Entandrophragma cylindricum) at the forest concession level in Cameroon

Illegal logging and associated trade are the cause of many economic and ecological problems both in producer and in consumer countries. There are an increasing number of national and international regulations in place that call for efficient timber tracking systems. We present results of a pilot study of a DNA-based method to control the geographical origin of timber in forest concessions in Cameroon. We addressed genetic differentiation at five nuclear microsatellite loci in seven sapelli (Entandrophragma cylindricum, Meliaceae) populations located in three forest concessions in Eastern Cameroon. In the framework of a blind test, seven anonymous timber sample sets were analysed at three microsatellite loci and compared to the genetic reference data of the forest concessions in Cameroon. Our results show that genetic differentiation was low within and among concessions. Combining the results of Bayesian genetic assignment method and exclusion test, we could determine that the timber stemmed or did not stem from the focus forest concession in six out of the seven blind sample sets. We further discuss the accuracy of the presented method and draw conclusions for a better sampling and genotyping strategy. Our work provides clear evidence that the use of genetic fingerprints is a useful tool to fight against illegal logging.

Development of DNA -based methods to identify CITES -protected timber species: a case study in the Meliaceae family

Abstract Violation of CITES regulations in tropical timber trade necessitates the development of accurate species identification systems. The application of available methods, mostly based on visual illustrations and descriptions of wood anatomical characteristics, proved to be difficult or even impossible, particularly on lower taxonomic levels. Further, because most of the chain-of-custody documents are externally applied marks which can easily be manipulated, control methods should be based on wood features which are inherent in the wood itself. In a case study on five closely-related genera of the Meliaceae (mahogany) family, including Swietenia sp. (listed on CITES appendix II), Khaya, Entandrophragma, and Carapa sp. (legal trade timbers), this study demonstrates the process of developing DNA markers for identification purposes. A detailed sequence analysis of several non-coding cpDNA regions resulted in an assay of seven genus-specific SNP (single nucleotide polymorphism) markers. Tools have been designed that could be applied with low-cost equipment on the basis of PCR-RFLPs without the need for sequencing or capillary electrophoresis techniques. In addition, the application of the method to wood material with degraded DNA of low overall quantity is highlighted.