Conservation biologists assign population distinctiveness by classifying populations as evolutionarily significant units (ESUs). Historically, this classification has included ecological and genetic data. However, recent ESU concepts, coupled with increasing availability of data on neutral genetic variation, have led to criteria based exclusively on molecular phylogenies. We argue that the earlier definitions of ESUs, which incorporated ecological data and genetic variation of adaptive significance, are more relevant for conservation. Furthermore, this dichotomous summary (ESU or not) of a continuum of population differentiation is not adequate for determining appropriate management actions. We argue for a broader categorization of population distinctiveness based on concepts of ecological and genetic exchangeability ( sensu Templeton).

Considering evolutionary processes in conservation biology.

The effect of Quaternary climatic changes on plant distribution and evolution

A compilation of studies using RAPD markers for evaluating population differentiation resulted in 78 estimates of AMOVA-derived Φ ST and 31 estimates of Nei's G ST , as well as in 41 estimates of Nei's within-population diversity. In outcrossing taxa, estimates of between-population diversity were closely correlated with maximum geographic distance between sampled populations. A corresponding association was not found in selfing taxa. These results suggest that RAPD can be a sensitive method for detection of genetic structuring according to the isolation-by-distance model. However, it also means that sampling strategies, as applied in individual studies, can seriously influence the resulting estimates of between-population diversity. Other sampling strategies, like number of plants per population and number of scored polymorphic markers, do not seem to impart any serious artefacts. As previously verified with allozyme data, RAPD markers showed that long-lived, outcrossing, late successional taxa retain most of their genetic variability within populations. By contrast, annual, selfing and/or early successional taxa allocate most of the genetic variability among populations. Estimates for between- and within-population diversity, respectively, proved to be negatively correlated, as previously reported for allozyme data. The only major discrepancy between allozymes and RAPD markers concerns geographic range; within-population diversity was strongly affected by distributional range of the investigated species in the allozyme data but not in the RAPD data. Moreover, RAPD-based values for between-population diversity increased with increasing distributional range whereas the opposite has been reported in a large allozyme data compilation. Contrary to allozymes, RAPD marker-derived within-population diversity is probably therefore not a very good predictor of total species genetic diversity.

Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants

The theoretical impacts of anthropogenic habitat degradation on genetic resources have been well articulated. Here we use a simulation approach to assess the magnitude of expected genetic change, and review 31 studies of 23 neotropical tree species to assess whether empirical case studies conform to theory. Major differences in the sensitivity of measures to detect the genetic health of degraded populations were obvious. Most studies employing genetic diversity (nine out of 13) found no significant consequences, yet most that assessed progeny inbreeding (six out of eight), reproductive output (seven out of 10) and fitness (all six) highlighted significant impacts. These observations are in line with theory, where inbreeding is observed immediately following impact, but genetic diversity is lost slowly over subsequent generations, which for trees may take decades. Studies also highlight the ecological, not just genetic, consequences of habitat degradation that can cause reduced seed set and progeny fitness. Unexpectedly, two studies examining pollen flow using paternity analysis highlight an extensive network of gene flow at smaller spatial scales (less than 10 km). Gene flow can thus mitigate against loss of genetic diversity and assist in long-term population viability, even in degraded landscapes. Unfortunately, the surveyed studies were too few and heterogeneous to examine concepts of population size thresholds and genetic resilience in relation to life history. Future suggested research priorities include undertaking integrated studies on a range of species in the same landscapes; better documentation of the extent and duration of impact; and most importantly, combining neutral marker, pollination dynamics, ecological consequences, and progeny fitness assessment within single studies.

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Genetic resource impacts of habitat loss and degradation; reconciling empirical evidence and predicted theory for neotropical trees

PREFACE REPETITIVE DNA: AN IMPORTANT SOURCE OF VARIATION IN EUKARYOTIC GENOMES Categories of DNA Sequence Mutations Tandem-Repetitive DNA: The Biology of Mini- and Microsatellites Minisatellites Microsatellites Transposable Elements Class I Transposons Class II Transposons Unclassified Transposons Transposons and Genome Evolution Transposons as Molecular Markers DETECTING DNA VARIATION BY MOLECULAR MARKERS Properties of Molecular Markers Traditional Marker Systems Protein Markers and Allozymes DNA Sequencing Restriction Fragment Length Polymorphism (RFLP) Analysis The PCR Generation: Molecular Markers Based on In Vitro DNA Amplification Principle of the PCR Cleaved Amplified Polymorphic Sequences PCR with Arbitrary Primers: RAPD and Its Variants Microsatellites Inter-Repeat PCR DNA Profiling of Genic Regions: Resistance Gene Analog Polymorphism, Sequence-Related Amplified Polymorphism, and Target Region Amplification Polymorphism Hybridization of Microsatellites to RAPD and MP-PCR Products AFLP Analysis and Its Variants Single-Strand Conformation Polymorphism Analysis and Related Techniques Miscellaneous Techniques LABORATORY EQUIPMENT METHODOLOGY Safety Precautions Isolation, Purification, and Quantitation of Plant DNA Collection and Preservation of Plant Tissue in the Field Plant DNA Extraction: General Considerations CTAB Protocol I CTAB Protocol II SDS-Potassium Acetate Protocol DNA Preparation via Nuclei Quantitation of DNA Basic Molecular Techniques Restriction of DNA Polymerase Chain Reaction DNA Sequencing Agarose Gel Electrophoresis PAA Gel Electrophoresis Detection of DNA in Gels Gel Drying Southern Blotting Generation of Radiolabeled Probes, Primers, and PCR Products Blot Hybridization Signal Detection PCR with Arbitrary Primers Standard RAPD Protocol Influence of Reaction Conditions and Components Modifications Microsatellite-Primed PCR Standard Protocol of Microsatellite-Primed PCR Influence of Reaction Conditions and Components Modifications PCR and Hybridization: Combinatory Techniques Assessing the Genomic Copy Number of PCR Amplicons Testing the Homology of Comigrating Bands Random Amplified Polymorphic Microsatellites Amplified Fragment Length Polymorphism Standard AFLP Protocol Using Radioisotopes AFLP Protocol Using Fluorescence-Labeled Primers Selective Amplification of Microsatellite Polymorphic Loci and Microsatellite AFLP Protocols Technical Aspects and Modifications Robustness and Reproducibility Generation and Analysis of Microsatellite Markers Microsatellite Analysis Using Radioisotopes Microsatellite Analysis Using Fluorochromes Technical Aspects and Modifications Generating Microsatellite Markers Without Cloning Microsatellite Cloning CAPS Analysis of cpDNA and mtDNA Standard CAPS Protocol Choice of CAPS Primers EVALUATION OF MOLECULAR MARKER DATA Robustness and Reproducibility Reliability Band Homology Band Linkage and Neutrality Fragment Sizing and Matching General Precautions Equipment Multilocus vs. Single-Locus Approaches Multilocus Markers Single-Locus Markers and Polyploids Band Sharing and Genetic Distances Coefficients of Similarity Dissimilarity Coefficients and Genetic Distances Identity and Uniqueness Clonal Structure Ordination, Clustering, and Dendrograms Ordination Techniques Construction of Dendrograms Population Genetic Analysis Measures of Variation Genetic Differentiation between Populations Genetic Distances between Populations Inbreeding Coefficient and Mating Systems Estimation of Relatedness and Paternity Testing Migration and Hybridization Gene Flow, Isolation-by-Distance, and Spatial Structure Phylogeography and Nested Clade Analysis Statistical Testing of Hypotheses: Analytical and Computational Methods APPLICATIONS OF DNA FINGERPRINTING IN PLANT SCIENCES A Brief History of DNA Fingerprinting Minisatellite and Oligonucleotide DNA Probes Detect Genetic Variation PCR-Based Methods Enter the Stage Microsatellite DNA Analyses Yield Codominant Markers Universal Organellar DNA Primers Produce Uniparental Markers Genotype Identification Individual-Specific DNA Fingerprints Cultivar Identification In Vitro-Propagated Plant Material and Somaclonal Variation Sports and Other Mutants Genetic Diversity Variation and Relatedness among Cultivars Analysis of Population Genetic Diversity and Its Distribution Hybridization and Introgression Plant Conservation Germplasm Characterization and Preservation Plant Taxonomy and Systematics Taxonomic Relationships Revealed by Multilocus DNA Methods Microsatellite Markers in Taxonomic Studies Taxonomic Consequences from DNA Profiling Data Phylogeography Phylogeography Based on cpDNA Phylogeography Based on Nuclear Genes LINKAGE ANALYSIS AND GENETIC MAPS Generating High-Density Genetic Maps Selection of Parent Plants Mapping Population Linkage Analysis The Genetic Map Cytogenetic Maps Genetic vs. Physical Maps Synteny: The Comparative Analysis of Genomes Marker-Assisted Selection Molecular Markers and Positional Cloning WHICH MARKER FOR WHAT PURPOSE: A COMPARISON Morphological Characters and Allozymes vs. DNA Markers Different Kinds of DNA Markers Discriminatory Power Genetic Distances Within- and Among-Population Variation Gene Tagging and Genetic Linkage Mapping Costs Conclusions FUTURE PROSPECTS: SNIPS AND CHIPS FOR DNA AND RNA PROFILING Single-Nucleotide Polymorphisms What Is a SNiP SNP Discovery DNA Microarrays Expression Profiling and Expression Markers APPENDIX 1: PLANT DNA ISOLATION PROTOCOLS APPENDIX 2: SUPPLIERS AND SELLERS OF REAGENTS AND EQUIPMENT APPENDIX 3: COMPUTER PROGRAMS DEALING WITH THE EVALUATION OF DNA SEQUENCE VARIATION AND MOLECULAR MARKER DATA APPENDIX 4: WEB PAGES OF INTEREST REFERENCES INDEX

DNA fingerprinting in plants: principles, methods, and applications

Tree species are becoming the focus of increasing conservation concern, with some 9000 species now threatened globally. Studies of intraspecific variation can contribute to the development of conservation strategies, by identifying appropriate units for conservation. The recent application of molecular techniques to a variety of tree species has highlighted a far higher degree of population differentiation than indicated by previous isozyme analyses, a result consistent with theoretical predictions. Analysis of the geographic distribution of cpDNA lineages has also enabled current patterns of population differentiation to be related to postglacial migration routes from different forest refugia. Such results highlight the importance of refugial areas for conservation of intraspecific variation in tree species.

Molecular phylogeography, intraspecific variation and the conservation of tree species

Cedrela odorata L. (Spanish cedar), an economically important timber species native to the American tropics, is the focus of increasing conservation concern due to high rates of deforestation within its native range. To assess the extent of the genetic diversity within and between populations of this species, samples were obtained from 10 widely dispersed populations within Costa Rica and analysed for random amplified polymorphic DNA (RAPD) variation. Fourteen 10-mer primers were used to generate 97 polymorphic RAPD bands. Presence/absence data for all bands were subjected to a pairwise genetic distance analysis, according to Jaccard's coefficient, then neighbour-joining cluster analysis was performed on these distances, as was an analysis of molecular variance (AMOVA), to assess levels of differentiation between populations and regions, and Shannon's Diversity Index was used to quantify levels of diversity within and between populations. Results indicated highly significant genetic differentiation (P < 0.004, AMOVA) between populations originating from the North Pacific and Atlantic/South Pacific regions of Costa Rica, with 35.3% of the total variation attributable to a difference between these areas. Little differentiation was recorded between populations from within the same region (P= 0.757, AMOVA), and 65.1% of the total variance was attributable to variation within populations. Estimated values for within-population diversity, calculated as Hpop/Hsp by means of Shannon's Diversity Index, were found to vary greatly between primers, but the overall within-population component of genetic diversity was 0.45. Possible reasons for the high degree of intraspecific genetic variation within this species are discussed and the implications of these results for the conservation and use of its genetic resources are described.

A. C. M. Gillies

Papers

Molecular phylogeography, intraspecific variation and the conservation of tree species

Genetic variation in Costa Rican populations of the tropical timber species Cedrela odorata L., assessed using RAPDs

Mahogany as a genetic resource

Mahogany as a genetic resource: Is there a future for mahogany?Edited by Andrew MacLellan