Richard A. Ennos

Estimating the relative rates of pollen and seed migration among plant populations

Interpopulation gene flow in plants is mediated by a combination of pollen and seed dispersal. The effectiveness of pollen and seeds in bringing about gene flow depends upon the mode of inheritance of the genetic marker. For nuclear and paternally inherited markers, gene flow occurs in both pollen and seed. For maternally inherited markers, genes are only dispersed in seeds. As a result, levels of population differentiation under drift-migration equilibrium are expected to differ for markers with contrasting modes of inheritance, and the extent of such differences should be related to the relative levels of pollen and seed migration among populations. A model is developed that relates expected values of population differentiation (Fst) for nuclear, paternally and maternally inherited markers, to pollen and seed migration rates. The model is used to estimate the relative rates of seed and pollen flow in six species of plants where Fst values are available for both nuclear and maternally inherited markers. Estimates of (pollen flow/seed flow) range from four in wild barley to 200 in oaks, and this pattern of variation is consistent with the reproductive characteristics of the species concerned.

Molecular phylogeography, intraspecific variation and the conservation of tree species

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.

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation.

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d) were detected. Within Spain all three major mitotypes were found, gene diversity was high, HT = 0.586, and this diversity was distributed predominantly among rather than within populations (FST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada. Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a; while populations in northern Fennoscandia were fixed for mitotype b. The isolated Scottish populations were predominantly of mitotype a, but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (HT = 0.120) and genetic differentiation among populations (FST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present‐day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.

Next-generation hybridization and introgression.

Hybridization has a major role in evolution—from the introgression of important phenotypic traits between species, to the creation of new species through hybrid speciation. Molecular studies of hybridization aim to understand the class of hybrids and the frequency of introgression, detect the signature of ancient hybridization, and understand the behaviour of introgressed loci in their new genomic background. This often involves a large investment in the design and application of molecular markers, leading to a compromise between the depth and breadth of genomic data. New techniques designed to assay a large sub-section of the genome, in association with next-generation sequencing (NGS) technologies, will allow genome-wide hybridization and introgression studies in organisms with no prior sequence data. These detailed genotypic data will unite the breadth of sampling of loci characteristic of population genetics with the depth of sequence information associated with molecular phylogenetics. In this review, we assess the theoretical and methodological constraints that limit our understanding of natural hybridization, and promote the use of NGS for detecting hybridization and introgression between non-model organisms. We also make recommendations for the ways in which emerging techniques, such as pooled barcoded amplicon sequencing and restriction site-associated DNA tags, should be used to overcome current limitations, and enhance our understanding of this evolutionary significant process.

Historical and contemporary mating patterns in remnant populations of the forest tree fraxinus excelsior L

Abstract Genetic variation at microsatellite markers was used to quantify genetic structure and mating behavior in a severely fragmented population of the wind‐pollinated, wind‐dispersed temperate tree Fraxinus excelsior in a deforested catchment in Scotland. Remnants maintain high levels of genetic diversity, comparable with those reported for continuous populations in southeastern Europe, and show low interpopulation differentiation (θ= 0.080), indicating that historical gene exchange has not been limited (Nm=3.48). We estimated from seeds collected from all trees producing fruits in three of five remnants that F. excelsior is predominantly outcrossing (tm = 0.971 ± 0.028). Use of a neighborhood model approach to describe the relative contribution of local and long‐distance pollen dispersal indicates that pollen gene flow into each of the three remnants is extensive (46–95%) and pollen dispersal has two components. The first is very localized and restricted to tens of meters around the mother trees. The second is a long distance component with dispersal occurring over several kilometers. Effective dispersal distances, accounting for the distance and directionality to mother trees of sampled pollen donors, average 328 m and are greater than values reported for a continuous population. These results suggest that the opening of the landscape facilitates airborne pollen movement and may alleviate the expected detrimental genetic effects of fragmentation.

Genetic variation in the vulnerable and endemic Monkey Puzzle tree, detected using RAPDs

Araucaria araucana (Monkey Puzzle), a southern South American tree species of exceptional cultural and economic importance, is of conservation concern owing to extensive historical clearance and current human pressures. Random amplified polymorphic DNA (RAPD) markers were used to characterise genetic heterogeneity within and among 13 populations of this species from throughout its natural range. Extensive genetic variability was detected and partitioned by analysis of molecular variance, with the majority of variation existing within populations (87.2%), but significant differentiation was recorded among populations (12.8%). Estimates of Shannons genetic diversity and percent polymorphism were relatively high for all populations and provide no evidence for a major reduction in genetic diversity from historical events, such as glaciation. All pairwise genetic distance values derived from analysis of molecular variance (ΦST) were significant when individual pairs of populations were compared. Although populations are geographically divided into Chilean Coastal, Chilean Andes and Argentinean regions, this grouping explained only 1.77% of the total variation. Within Andean groups there was evidence of a trend of genetic distance with increasing latitude, and clustering of populations across the Andes, suggesting postglacial migration routes from multiple refugia. Implications of these results for the conservation and use of the genetic resource of this species are discussed.

Paternity analysis of pollen-mediated gene flow for Fraxinus excelsior L. in a chronically fragmented landscape

Paternity analysis based on microsatellite marker genotyping was used to infer contemporary genetic connectivity by pollen of three population remnants of the wind-pollinated, wind-dispersed tree Fraxinus excelsior, in a deforested Scottish landscape. By deterministically accounting for genotyping error and comparing a range of assignment methods, individual-based paternity assignments were used to derive population-level estimates of gene flow. Pollen immigration into a 300 ha landscape represents between 43 and 68% of effective pollination, mostly depending on assignment method. Individual male reproductive success is unequal, with 31 of 48 trees fertilizing one seed or more, but only three trees fertilizing more than ten seeds. Spatial analysis suggests a fat-tailed pollen dispersal curve with 85% of detected pollination occurring within 100 m, and 15% spreading between 300 and 1900 m from the source. Identification of immigrating pollen sourced from two neighbouring remnants indicates further effective dispersal at 2900 m. Pollen exchange among remnants is driven by population size rather than geographic distance, with larger remnants acting predominantly as pollen donors, and smaller remnants as pollen recipients. Enhanced wind dispersal of pollen in a barren landscape ensures that the seed produced within the catchment includes genetic material from a wide geographic area. However, gene flow estimates based on analysis of non-dispersed seeds were shown to underestimate realized gene immigration into the remnants by a factor of two suggesting that predictive landscape conservation requires integrated estimates of post-recruitment gene flow occurring via both pollen and seed.

Chloroplast DNA diversity in Calluna vulgaris (heather) populations in Europe

Variation in the chloroplast genome of Calluna vulgaris (heather), the dominant species of northwest European heath communities, was analysed using polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) and microsatellites. No length polymorphisms were detected in the 100–200 base pair (bp) fragments amplified by the conserved microsatellite primers, and sequencing revealed that the repeat regions were interrupted relative to the corresponding sequence in Nicotiana tabacum. In contrast, PCR–RFLP analysis revealed high levels of haplotype diversity within populations (hS = 0.443, hT = 0.842), as well as substantial differentiation between populations (GST = 0.473). Diversity and differentiation were higher in southern Europe than in northern Europe. Interpreted in the light of data from allozyme studies and pollen core records, the results suggest that the main glacial refugia for C. vulgaris were located in southwest Europe, including northern Spain, the Pyrenees and the Massif Central region of France. There is also evidence for diffuse survival of the species at more northerly latitudes.

Identifying the early genetic consequences of habitat degradation in a highly threatened tropical conifer, Araucaria nemorosa Laubenfels

The early genetic effects of habitat degradation were investigated in the critically endangered conifer Araucaria nemorosa. This species occurs in New Caledonia, a global biodiversity hotspot where the worlds greatest concentration of endemic conifer species coincides with an extremely high level of habitat destruction due to fire and mining. Using seven microsatellite loci, estimates were made of genetic marker variation, inbreeding coefficients and population differentiation of adult and seedling cohorts of A. nemorosa. These were contrasted with equivalent estimates, made over similar spatial scales and with the same marker loci, in the locally common and more widespread sister species Araucaria columnaris. There were no significant differences in population genetic parameters between adult populations of the two species, despite their different abundances. However, in A. nemorosa, the juvenile cohort showed a loss of rare alleles and elevated levels of inbreeding when compared to the adult cohort. These genetic differences between the cohorts were not observed in the locally common A. columnaris. This suggests that recent environmental degradation is influencing the genetic structure of A. nemorosa populations. Although this is not detectable among predisturbance adult populations, an early warning of these impacts is evident in more recently established seedling cohorts. The conservation implications of these results are discussed.

Neutral DNA markers fail to detect genetic divergence in an ecologically important trait

The development of strategies for in situ, ex situ conservation and reforestation of the monkey puzzle tree (Araucaria araucana), a vulnerable tree endemic to southern South America, has led to an interest in the level and distribution of the genetic diversity of the species. Neutral DNA markers (RAPDs) and quantitative genetic techniques were used to characterise genetic heterogeneity within and among populations from throughout the natural range of the species. Both the level and pattern of genetic variation estimated using the different techniques were essentially uncorrelated. An important discrepancy was found with the neutral markers failing to detect an important quantitative genetic divergence across the Andean Range relating to drought tolerance. This study clearly demonstrates the potential problems associated with making recommendations for conserving the genetic resource of threatened species based solely on neutral marker studies. Alternative approaches are discussed, including a stronger focus on ecologically important traits and the potential use of surrogate measures of genetic variability.