Pollen, wind and fire: how to investigate genetic effects of disturbance-induced change in forest trees.

Understanding the consequences of habitat disturbance on mating patterns although pollen and seed dispersal in forest trees has been a long-standing theme of forest and conservation genetics. Forest ecosystems face global environmental pressures from timber exploitation to genetic pollution and climate change, and it is therefore essential to comprehend how disturbances may alter the dispersal of genes and their establishment in tree populations in order to formulate relevant recommendations for sustainable resource management practices and realistic predictions of potential adaptation to climate change by means of range shift or expansion (Kremer et al. 2012). However, obtaining reliable evidence of disturbance-induced effects on gene dispersal processes from empirical evaluation of forest tree populations is difficult. Indeed, tree species share characteristics such as high longevity, long generation time and large reproductive population size, which may impede the experimenter's ability to assess parameters at the spatial and time scales at which any change may occur (Petit and Hampe 2006). It has been suggested that appropriate study designs should encompass comparison of populations before and after disturbance as well as account for demonstrated variation in conspecific density, that is, the spatial distribution of mates, and forest density, including all species and relating to alteration in landscape openness (Bacles & Jump 2011). However, more often than not, empirical studies aiming to assess the consequences of habitat disturbance on genetic processes in tree populations assume rather than quantify a change in tree densities in forests under disturbance and generally fail to account for population history, which may lead to inappropriate interpretation of a causal relationship between population genetic structure and habitat disturbance due to effects of unmonitored confounding variables (Gauzere et al. 2013). In this issue, Shohami and Nathan (2014) take advantage of the distinctive features of the fire-adapted wind-pollinated Aleppo pine Pinus halepensis (Fig. 1) to provide an elegant example of best practice. Thanks to long-term monitoring of the study site, a natural stand in Israel, Shohami and Nathan witnessed the direct impact of habitat disturbance, here taking the shape of fire, on conspecific and forest densities and compared pre- and postdisturbance mating patterns estimated from cones of different ages sampled on the same surviving maternal individuals (Fig. 2). This excellent study design is all the more strong that Shohami and Nathan took further analytical steps to account for confounding variables, such as historical population genetic structure and possible interannual variation in wind conditions, thus giving high credibility to their findings of unequivocal fire-induced alteration of mating patterns in P. halepensis. Most notably, the authors found, at the pollen pool level, a disruption of local genetic structure which, furthermore, they were able to attribute explicitly to enhanced pollen-mediated gene immigration into the low-density fire-disturbed stand. This cleverly designed research provides a model approach to be followed if we are to advance our understanding of disturbance-induced dispersal and genetic change in forest trees.

Two are better than one: combining landscape genomics and common gardens for detecting local adaptation in forest trees.

Predicting likely species responses to an alteration of their local environment is key to decision-making in resource management, ecosystem restoration and biodiversity conservation practice in the face of global human-induced habitat disturbance. This is especially true for forest trees which are a dominant life form on Earth and play a central role in supporting diverse communities and structuring a wide range of ecosystems. In Europe, it is expected that most forest tree species will not be able to migrate North fast enough to follow the estimated temperature isocline shift given current predictions for rapid climate warming. In this context, a topical question for forest genetics research is to quantify the ability for tree species to adapt locally to strongly altered environmental conditions (Kremer et al. ). Identifying environmental factors driving local adaptation is, however, a major challenge for evolutionary biology and ecology in general but is particularly difficult in trees given their large individual and population size and long generation time. Empirical evaluation of local adaptation in trees has traditionally relied on fastidious long-term common garden experiments (provenance trials) now supplemented by reference genome sequence analysis for a handful of economically valuable species. However, such resources have been lacking for most tree species despite their ecological importance in supporting whole ecosystems. In this issue of Molecular Ecology, De Kort et al. () provide original and convincing empirical evidence of local adaptation to temperature in black alder, Alnus glutinosa L. Gaertn, a surprisingly understudied keystone species supporting riparian ecosystems. Here, De Kort et al. () use an innovative empirical approach complementing state-of-the-art landscape genomics analysis of A. glutinosa populations sampled in natura across a regional climate gradient with phenotypic trait assessment in a common garden experiment (Fig. ). By combining the two methods, De Kort et al. () were able to detect unequivocal association between temperature and phenotypic traits such as leaf size as well as with genetic loci putatively under divergent selection for temperature. The research by De Kort et al. () provides valuable insight into adaptive response to temperature variation for an ecologically important species and demonstrates the usefulness of an integrated approach for empirical evaluation of local adaptation in nonmodel species (Sork et al. ).

Thirteen microsatellites developed by SSR-enriched pyrosequencing for Solanum rostratum (Solanaceae) and related species.

PREMISE OF THE STUDY: Microsatellite markers were developed using second-generation sequencing in Solanum rostratum as a tool to study the reproductive biology and genetic structure of this invasive species. METHODS AND RESULTS: Thirteen microsatellites were successfully discovered and amplified in a single multiplexed PCR. All loci showed genetic variation in S. rostratum. Cross-amplification in five closely related taxa was successful for a subset of loci. CONCLUSIONS: The set of 13 microsatellite markers developed here provides a time-effective and cost-effective genetic tool to study the reproductive biology of S. rostratum. The demonstrated transferability of the PCR multiplex to related taxa also highlights its usefulness for evolutionary studies across Solanum sect. Androceras.

Fast sequence-based microsatellite genotyping development workflow.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20-40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

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

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 (E = 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 (t(m) = 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.

Comparison of random and SSR-enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization in Acacia harpophylla F. Muell. Ex Benth.

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state-of-the-art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north-eastern Australia, we show that high-throughput shotgun pyrosequencing technology, so-called second-generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large-scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200,908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost-effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.

Taking a tree's perspective on forest fragmentation genetics.

Despite longstanding research, how anthropogenic disturbance affects the genetics of tree populations remains poorly understood. Although empirical evidence often conflicts with theoretical expectations, little progress has been made in refining experimental design or in reformulating theoretical hypotheses. Such progress is, however, essential to understand how forest tree species can tolerate anthropogenic disturbance. Further advances in forest fragmentation genetics research will require that processes driving reproduction and recruitment in fragmented populations are assessed from a tree's perspective instead of experimental convenience, using a multidisciplinary approach to explain the spatiotemporal dynamics of gene dispersal. In this opinion article we aim to inspire a new perspective in forest fragmentation genetics research.

Effective seed dispersal across a fragmented landscape.

The role of seed dispersal in maintaining genetic connectivity among forest fragments has largely been ignored because gene flow by pollen is expected to predominate. By using genealogical reconstruction, we investigated gene flow after establishment of seeds in a wind-pollinated, wind-dispersed tree. Our data show that seed dispersal is the main vector of gene flow among remnants and that long-distance dispersal is common across a chronically fragmented landscape. The relative importance of seed-mediated gene flow may have been underemphasized in other fragmented systems, and diagnosing the response of forest trees to current anthropogenic disturbances requires the assessment of phenomena after establishment.