Management strategies for coral reefs are dependant on information about the spatial population structure and connectivity of reef organisms. Genetic tools can reveal important information about population structure, however, this information is lacking for many reef species. We used a mitochondrial molecular marker to examine the population genetic structure and the potential for meta-population dynamics in a direct developing coral reef fish using 283 individuals from 15 reefs on the Great Barrier Reef, Australia. We employed a hierarchical sampling design to test genetic models of population structure at multiple geographical scales including among regions, among shelf position and reefs within regions. Predictions from island, isolation-by-distance and meta-population models, including the potential for asymmetric migration, local extinction and patterns of re-colonisation were examined.Acanthochromis polyacanthus displayed strong genetic structure among regions  that supported an equilibrium isolation-by-distance model . Significant structuring across the continental shelf was only evident in the northern region  and no evidence of isolation-by-distance was found within any region. Pairwise ΦST values indicated overall strong but variable genetic structure , and asymmetric migration rates among reefs with low genetic structure. Genetic differentiation among younger reefs was greater than among older reefs supporting a meta-population propagule-pool colonisation model. Variation in genetic diversities, demographic expansion and population growth estimates indicated more frequent genetic bottlenecks/founder effects and subsequent population expansion in the central and southern regions compared to the northern one.Our findings provide genetic evidence for meta-population dynamics in a direct developing coral reef fish and we reject the equilibrium island and isolation-by distance models at local spatial scales. Instead, strong non-equilibrium genetic structure appears to be generated by genetic bottlenecks/founder effects associated with population reductions/extinctions and asymmetric migration/(re)-colonisation of such populations. These meta-population dynamics varied across the geographical range examined with edge populations exhibiting lower genetic diversities and higher rates of population expansion than more central populations. Therefore, coral reef species may experience local population reductions/extinctions that promote overall meta-population genetic differentiation. Coral reefs are important ecosystems in ecological, evolutionary and socio-economic contexts but are under increasing threat from anthropogenic impacts ,2. The mGenetic models of spatial structure have developed from Wright's original island model  into theIn contrast to these island-based models, meta-population theory attempts to understand systems of evolutionarily ephemeral, genetically subdivided populations that persist through time via colonisation and migration from source populations -23. SuchTheory suggests that the sources and rates of colonisation relative to subsequent migration are critical determinants of the evolution of the genetic structure of meta-populations ,19,23. IThe effects of these meta-population dynamics on spatial genetic structure have typically been estimated in terms of genetic differentiation among populations, but may also be evident in patterns of genetic diversity and demographic history of local populations ,40,41. AFishes on coral reefs occupy a naturally fragmented environment where patches of suitable reef habitat are surrounded by unsuitable habitat such as open sand and deep water making them amenable to analysis under a meta-population framework. At present, however, we know little about the presence, spatial extent and genetic consequences of meta-populations dynamics in marine systems , but see]. SpecieAcanthochromis polyacanthus is a common fish on Australia's Great Barrier Reef (GBR) and lacks a dispersive larval phase. This life-history trait, coupled with the physical history of the GBR, and the sensitivity of mitochondrial molecular markers to drift, provides an opportunity to evaluate the potential importance of meta-population dynamics to the evolution of genetic structure on small spatial scales in a natural marine system. Previous investigations of A. polyacanthus, as well as the presence of several colour morphs on the GBR, suggest that sufficient time has elapsed since colonisation of the GBR began by this species for it to have evolved genetic differences among populations separated by small geographic distances  a,71 aST ves . Fur. FurA. pA. polyacanthus were collected from 15 back-reefs from 3 regions on the Great Barrier Reef during 2003 and 2004  were taken and preserved in 80% EtOH. Genetic effects of continental shelf position, inner, middle and outer, were examined among replicate reefs in two regions . Because the southern region contains no true inner- and mid-shelf zones, the genetic structure in this region was explored using pairwise genetic distances2 of fin tissue  using a modified CTAB extraction procedure .Genomic DNA was extracted from approx 0.25 cmocedure [, excludiocedure [. PCR reaThe control region sequences were aligned and edited using Sequencher 4.2 (GeneCodes Corp. Michigan USA) and ESEE . The besEstimates of mitochondrial haplotype and nucleotide diversity -93 and tA. polyacanthus among regions and reefs was explored using AMOVA with 1000 permutations [Hierarchical population genetic structure of utations ,96 impleutations .em from a to b, and vice versa) using a coalescence maximum likelihood approach (Markov Chain Monte Carlo with Hastings Metropolis importance sampling) and assumes constant mutation rates and equal effective population sizes. Because of the molecular divergences detected by phylogenetic and AMOVA analyses, Migrate was run on each geographical region separately. Reciprocal migration rates were interpreted as different when their 95% confidence intervals did not overlap. Extensive sampling regimes including 10 short chains sampled 10,000 times each and 5 long chains sampled 100,000 each were averaged over 5 replicates. Migrate was implemented on a SGI Origin 3800 computer in the James Cook University High Performance Computing Facility using a ts/tv ratio of 1.53 (estimated by Modeltest). Earlier versions of Migrate had problems with convergence of estimates, migration estimates and their associated profile likelihoods in low signal data [Differences in levels of migration among reefs were investigated further using Migrate 1.7.6.1 ,34. Thisnal data . Here weST, and geographical distances among locations were calculated using Vincenty's inverse method http://www.ga.gov.au/nmd/geodesy/datums/distance.jsp. Correlations between genetic and geographical distances were tested using a Mantel test  of both log-transformed and non-transformed data following [ST are presented here.Genetic distances were estimated using the conventional genetic distance estimator, Φollowing  and implollowing . A falseollowing . Log traDemographic histories were explored using mismatch analysis in Arlequin and DnaSP  using 10g) was calculated among reefs and regions using a maximum likelihood coalescence approach implemented in Fluctuate 1.4 [th step, followed by ten long chains each of 20000 steps sampled every 20th step, gave consistent results among runs and was used in all analyses. Estimates of g were compared among regions using a Kruskal-Wallis test. We calculated Fu's Fs [2 [The exponential population growth parameter .Pairwise genetic distances among reef samples. Pairwise ΦClick here for fileA. polyacanthus was explored using Bayesian inference implemented in MrBayes 3.0B4 [Amphiprion melanopus and A. akindynos. Credibility values were obtained from a majority rule consensus tree of the last 2000 trees and values greater than 90% are indicated on the major nodes of the tree.Tree of unique haplotypes. The phylogenetic structure of es 3.0B4 . The anaClick here for file


Substantial decreases in mtDNA variation between time points were observed in populations from just two islands (Marchena and Genovesa). Our results suggests that, for the majority of islands, a single, intense El Niño event did not reduce marine iguana populations to the point where substantial neutral genetic diversity was lost. In the case of Marchena, simultaneous changes to both nuclear and mitochondrial DNA variation may also be the result of a volcanic eruption on the island in 1991. Therefore, studies that seek to evaluate the genetic impact of El Niño must also consider the confounding or potentially synergistic effect of other environmental and biological forces shaping populations.The El Niño-Southern Oscillation (ENSO) is a major source of climatic disturbance, impacting the dynamics of ecosystems worldwide. Recent models predict that human-generated rises in green-house gas levels will cause an increase in the strength and frequency of El Niño warming events in the next several decades, highlighting the need to understand the potential biological consequences of increased ENSO activity. Studies have focused on the ecological and demographic implications of El Niño in a range of organisms, but there have been few systematic attempts to measure the impact of these processes on genetic diversity in populations. Here, we evaluate whether the 1997–1998 El Niño altered the genetic composition of Galápagos marine iguana populations from eleven islands, some of which experienced mortality rates of up to 90% as a result of El Niño warming. Specifically, we measured the temporal variation in microsatellite allele frequencies and mitochondrial DNA diversity (mtDNA) in samples collected before (1991/1993) and after (2004) the El Niño event. Based on microsatellite data, only one island (Marchena) showed signatures of a genetic bottleneck, where the harmonic mean of the effective population size ( The El Niño-Southern Oscillation (ENSO) refers to a complex set of ocean-atmosphere interactions that take place throughout the Pacific basin During periods of acute El Niño warming, there is widespread mortality in aquatic organisms in the eastern Pacific Amblyrhynchus cristatus), a species that is only found in the archipelago. Marine iguanas are known to inhabit the shoreline of all the major islands in Galápagos, where they forage almost exclusively on algae from intertidal and (nearshore) subtidal zones. The digestion of algae is made possible by a community of bacterial micro-symbionts that exist in the hindgut of the iguanas The Galápagos archipelago  lies in Sharp population declines, or bottlenecks, like those seen during the recent El Niños, may translate into losses of genetic variation that can lead to increased rates of inbreeding and the fixation of deleterious alleles, and hinder the ability of populations to adapt to future changes in the environment eN) from such data, and methods based on demographic information tend to overestimate eN. Conversely, indirect methods of eN estimation based on genetic data have been shown to be particularly informative, making them an important component in evaluating the consequences of potential bottleneck events such as El Niño Although long-term demographic studies from several islands in Galápagos reveal the drastic effects of El Niño on marine iguana census numbers and reproductive behaviour, it is difficult to estimate the effective population size  and at least one generation after (2004) the warming event. In order to corroborate our results, we also analysed the complete mitochondrial control region for the same populations and most individuals for signs of temporal genetic changes. The large number of individuals and genetic loci used in our analysis, combined with the temporal sampling design, makes this one of the most comprehensive studies to date trying to measure the short term genetic effects of a severe El Niño event on natural populations.In this study, we estimated the harmonic mean of Approximately 800 marine iguanas from 11 islands in the Galápagos archipelago were sampled at two different time points before (in 1991/1993) and after (in 2004) the 1997–1998 El Niño event (see eN) by measuring the temporal variance of microsatellite loci allele frequencies between time points using a likelihood-based approach. For the analysis of temporal changes in allele frequencies for eN estimation, the program NeESTIMATOR requires the user to define a reference time point of a population (generation 0) and at least one subsequent generation (in our case generation 1). Island populations sampled in 1991/1993 were entered as generation 0 and those sampled in 2004 as generation 1. Observed changes in allele frequencies and therefore estimates of eN only reflect changes that occurred from the reference point (generation 0 in 1991/1993) to the subsequent point (generation 1 in 2004). Since the TM3 program requires a maximum value for eN, we used the estimates of the maximum census size (cN) of a respective island population eN, as in most published studies, cN exceeds or equals eNeN was then estimated as the maximum likelihood value of 10,000 updates within that range.Thirteen species-specific microsatellite loci were amplified in 806 individuals and scored for alleles as previously described ST differentiation between pre- and post-El Niño samplings was estimated with the program ARLEQUIN version 3.0 Ft-test described by Nei ST values and their significance were calculated between time points for each population using ARLEQUIN version 3.0 H-test η) and θ . From this test, p values were obtained which actually reflect where the observed Hd value falls on the simulated distribution. Although the H-test was originally based on the number of segregating sites in a population (S), DNASP uses η in place of S. We chose to run the additional set of simulations with θ because it should be less sensitive to low frequency migrants, which can greatly elevate the value of η, and give a false signal of lower than expected Hd.Complete mitochondrial CR sequences (1183 base pairs) were generated for 838 marine iguanas . PCR was carried out on total genomic DNA using the primers IguanaCytb3 (5′-ACCAGTAGAACACCCMTTCATC-3′) and 12s1984 ST values calculated between pre- and post-El Niño samples were not significantly different from zero for most comparisons, but was 0.008 and significant (p<0.05) for Marchena (ST differentiation (0.008) was also found between time point samplings on Floreana, but in this case it was not significant at the 5% level.The average heterozygosity of the 13 microsatellite loci calculated for each population and time point ranged from 0.637 for Pinta in 1993 to 0.815 for Fernandina in 2004. Island specific FMarchena . The samp-values in p = 0.032) from 1993 to 2004, while there was a significant increase (p = 0.04) on Santa Cruz from 1991 to 2004.Based on the Wilcoxon signed-ranks test, locus specific heterozygosity was not significantly different (α = 0.05) between pre- and post-El Niño samples from a specific island, with the exception of Marchena and Santa Cruz  obtained from microsatellite data using the temporal variance method. eN values for each population represent the harmonic mean of the effective population size over the time interval between pre- and post-El Niño samplings. A low eN value  was only found for the population on Marchena, with a eN around 40 individuals and a narrow 95% CI of 21–86 individuals. For the remaining islands, eN estimates ranged from 770 and 783 individuals on the islands of Floreana and Genovesa respectively, to as high as 120,000 individuals on Fernandina. However, with the exception of Marchena, eN estimates from other islands had large CI values that overlapped with the minimum and maximum census size numbers of a specific island population eN estimate of around 40 individuals  between 1993 and 2004 experienced by the population on Marchena , and are therefore in line with the FST results from the microsatellite data.Mitochondrial control region (CR) data was generated for nearly the same set of individuals and populations used in the microsatellite analysis see ; Table 2H-test) show that only Hd values for Marchena and Genovesa were significantly lower than expected under neutral evolution in samples from 2004 but not in those from 1991/1993, supporting the occurrence of some severe demographic event between time points (H-test description). Several other populations do show lower than expected levels of haplotype diversity compared to neutral expectations, but only for the first time point  or for both time points (Santiago), which does not indicate that a genetic bottleneck occurred between time samplings. Only a single CR haplotype was found in samples collected on Santa Cruz in both 1991 and 2004, and therefore genetic diversity analyses and simulations could not be carried out for this island population.Coalescent simulations of haplotype diversity  and at least 30 individuals per population eN beyond that of the simulation.The temporal sampling of genetic data can be a powerful means of estimating eN≤50) during the 1997–1998 warming period, it is very likely that iguanas sampled in 2004 would primarily be individuals born in the first generation after the bottleneck event and would therefore reflect the gene pool of the reduced population. On the other hand, if the population size remained fairly high during the warming period, the gene pool reflected in 2004 would not deviate significantly from its pre-El Niño composition. In the first scenario (eN≤50), given the number and variability of microsatellite loci employed in this study, we would be able to detect a critical reduction of eN with a rather high probability as pointed out above. In the second case, however, we would not expect to see any major changes in allele frequencies between time points, since the population makeup had not been significantly altered.In addition, the temporal variance approach, as well as other tests comparing populations before and after a population reduction , require that a population be sampled before and at least one generation after the potential bottleneck event. In our study, we collected marine iguana specimens from eleven different islands in 1991 or 1993, before the 1997–1998 El Niño, and in 2004, approximately six years after the end of the warming period. Since we sampled mainly adults and sub-adults in 2004, along with some juveniles, it is possible that some of these individuals were born prior to the El Niño period. However, if a given population was reduced to a critically small size  populations along the Chilean coast fell by 50% compared with unaffected populations following the 1982–1983 El Niño S. mendiculus), whose populations have been severely diminished by ENSO, have significantly lower observed heterozygosity compared to the common and unaffected Magellanic penguin (Spheniscus magellanicus) from South America, possibly the outcome of a series of strong El Niño events Arhopala epimuta) in Borneo found that El Niño-induced forest fires did not alter the temporal genetic structure of microsatellite loci (5 loci) or mtDNA (control region) in populations that were sampled before and after the 1997–1998 El Niño There have only been a few attempts to measure the impact of severe El Niño events on genetic diversity in natural populations, with mixed results. Randomly amplified polymorphic DNA (RAPD) markers have shown that heterozygosity in intertidal kelp  during intense El Niño periods and 2004 , indicatMarchena . Lastly,Marchena .The results of the mtDNA data were similar, where Marchena exhibited the largest decrease in haplotype diversity and nucleotide diversity from 1993 to 2004 ; Table 2The genetic signature of a population decline detected between 1991 and 2004 on Genovesa likely reflects the population crash, from 15,000 to 900 individuals, that was observed during the 1997–1998 El Niño event, and there is no evidence for other incidents which may have drastically effected the demography of this island during this time period [14 and Martin Wikelski pers. comm.]. Conversely, for Marchena, marine iguanas may also have been highly impacted by a strong volcanic eruption in 1991, where lava flows continued for at least 40 days. This eruption is likely to have caused mortality of marine iguanas in both the terrestrial environment  per generation. Thus, genetic diversity is lost more rapidly in small populations. Yet, during brief bottleneck events, only a small amount of the total heterozygosity is lost, even after a considerable reduction in population size (e.g. 2% after the first generation of a bottleneck of eN = 50) eN. Therefore, it is not surprising that, with the exception of Marchena, a single, strong El Niño event did not stimulate a loss of heterozygosity in the populations, even if mortality rates were high. The temporal variance approach, on the other hand, relies on shifts in allele frequencies rather than loss of diversity, and has been shown to be particularly sensitive in estimating eN in small populations eN estimates suggest that the effective size of most populations of marine iguanas were so large that they underwent only slight shifts in allele frequencies. Such temporal stability has been observed in a number of other studies Under random mating, heterozygosity is lost from a population at a rate of 1/2eN estimate for Marchena, calculated over an interval in which a severe El Niño event and a volcanic eruption occurred, suggests that it is important to consider the potential synergistic relationship between El Niño and other phenomena. While we saw little evidence of a bottleneck in the majority of our populations, future experimental designs must account for multiple natural  and human-induced (e.g. oil spill) disturbances, or else the causes of population reductions may be confounded.Although we did not find a strong influence of El Niño on genetic diversity in marine iguanas, the data show that the genetic impact of a single, intense environmental challenge may vary even within a single species and depends on the specific history of a population. The low eN over many generations eN is heavily influenced by generations in which the effective population size is small. Therefore, the high mortality rates of marine iguanas associated with El Niño warming may compound over multiple ENSO cycles. Since climate models predict that the strength and frequency of El Niño events will continue to increase, a true understanding of the long-term impact of ENSO on population persistence may only come from experiments designed to measure the genetic impact of this phenomenon following a series of consecutive events.Evolutionary theory predicts that the long-term effective size of a population is the harmonic mean of Table S1Sampling localities by island and sample sizes for 13 microsatellite loci and mitochondrial control region sequences (1183 bp) for the two temporal samplings (1991/1993 and 2004). The first column lists sampling localities by island, specific sampling location (in parentheses), and geographical coordinates. Sample sizes for 13 microsatellite loci and mitochondrial control region sequences (1183 bp) are reported in separate columns for the two temporal samplings before the 1997–1998 El Niño  and after the 1997–1998 El Niño in the year 2004.(0.04 MB DOC)Click here for additional data file.Table S2Locus specific heterozygosity values for 13 microsatellite loci for pre- and post-El Niño island samplings. Locus specific heterozygosities calculated using the program ARLEQUIN for marine iguana populations sampled before the 1997–1998 El Niño (in 1991 or in 1993 or as for Santa Fé in both years) and after the 1997–1998 El Niño in the year 2004. The first column (locus) shows names of microsatellite loci. The other columns report the heterozygosity for a given population for each time point. Differences in heterozygosity between time points were tested with a Wilcoxon signed ranks test and associated p-values are provided in the last line. Significant p-values (p<0.05) are marked with an asterik (*). The only significant decrease of locus specific heterozygosity from 1993 to 2004 was found for the population on Marchena, whereas Santa Cruz showed a significant increase during this period.(0.11 MB DOC)Click here for additional data file.Table S3List of haplotype diversity (Hd) values and their variance (V) for marine iguana populations. List of haplotype diversity (Hd) values and their variance (V) for marine iguana populations from both time points. T-values and their corresponding probabilities are based on the test adapted by Nei (0.04 MB DOC)Click here for additional data file.Table S4List of nucleotide diversity (π) values and their variance (V) for marine iguana populations. List of nucleotide diversity (π) values and their variance (V) for marine iguana populations from both time points. T-values and their corresponding probabilities are based on the test adapted by Nei (0.04 MB DOC)Click here for additional data file.Figure S1Graphical display of the TM3 results  as provided by the program NeESTIMATOR. Graphical display of the TM3 results (estimates of the effective population size (Ne) generated from the temporal variance of allele frequencies using a likelihood-based approach) as provided by the program NeESTIMATOR see . Point e(2.03 MB DOC)Click here for additional data file.


Forest trees are the dominant species in many parts of the world and predicting how they might respond to climate change is a vital global concern. Trees are capable of long-distance gene flow, which can promote adaptive evolution in novel environments by increasing genetic variation for fitness. It is unclear, however, if this can compensate for maladaptive effects of gene flow and for the long-generation times of trees. We critically review data on the extent of long-distance gene flow and summarise theory that allows us to predict evolutionary responses of trees to climate change. Estimates of long-distance gene flow based both on direct observations and on genetic methods provide evidence that genes can move over spatial scales larger than habitat shifts predicted under climate change within one generation. Both theoretical and empirical data suggest that the positive effects of gene flow on adaptation may dominate in many instances. The balance of positive to negative consequences of gene flow may, however, differ for leading edge, core and rear sections of forest distributions. We propose future experimental and theoretical research that would better integrate dispersal biology with evolutionary quantitative genetics and improve predictions of tree responses to climate change. The overall picture is a negative correlation between Δ and the latitude of origin of the population as shown in panel (b) by the example in the case of Pinus contorta nditions . Reactiocontorta . FiguresTrees are characterised by their particular life history, combining long-generation time  and the capacity for long dispersal distances through pollen and seeds. Given the anticipated intensity and directionality of climatic change, do trees have the adaptive capacity to respond and how will gene flow affect that response? Valuable insights into these issues are provided by the retrospective analysis of adaptation in tree populations during the climate change that occurred following the last glaciations  gene flow could compensate for the long-generation time of trees, facilitating evolutionary change in a shifting climate, by reviewing both data and theory, about the extent of gene flow in forest trees and its evolutionary consequences. We first review the recent literature on LD pollen and seed dispersal in trees and show that their spatial range is frequently comparable to the predicted scale of habitat shifts within one generation. We then review the theoretical predictions and experimental evidence for the effects that such LD gene flow could have on local adaptation in trees. We conclude that the positive effects of gene flow may often dominate its negative effects, although regional variation may influence the balance of those effects. We finally elaborate on the theoretical and experimental approaches that should be implemented to improve our ability to predict the scale and distribution of gene flow effects on forest ecosystems in the context of climate change.effective propagule dispersal (Glossary) in trees depends on a variety of physical and biological processes that determine the amount and availability of pollen and seeds, their movement, their viability before and during movement, and the probability of successful pollination leading to viable seed and the seedling establishment rates. Different combinations of these components may yield effective dispersal (Glossary) distances spanning from a few centimetres to thousands of kilometres . Interactions between physical and biological processes influencing LD dispersal (LDD) and the way these will be affected in a changing climate are particularly poorly understood. The local environment around the release point of the propagule and the conditions at which the propagule was released can have strong effects of either decreasing or increasing the rate and extent of LD propagule transport  between pollen release and fertilisation. Similarly, quantifying the multiple demographic and dispersal parameters affecting seed dispersal and subsequent establishment is still hard to accomplish  dates along temperature gradients within the range. The simple conceptual model in genetic load (Glossary) depressing population mean fitness, and thus population growth , θ is the optimal phenotype in that location, P is the local phenotypic variance around this mean. This expression shows that the mean fitness in a variable population subject to stabilising selection is reduced in two ways: (1) Standing load (P/2Vs): caused by phenotypic variation and present even when the mean phenotype matches the optimum; (2) Evolutionary load [Evolutionary loads can be generated by selection that varies over space or time ; this part of the load increases with dispersal distance (σ). The second term describes phenotypic mismatch due to the lagging response of the mean phenotype to temporal change in the local optimum (lag load). This part of the load decreases with dispersal distance because migration helps the species track its shifting optimum through space. The third term in eqn 2.2 shows that the evolutionary load declines with genetic variance G because response to selection increases. Dispersal distance also affects the evolution of genetic variance G (evolutionary load (eqn 2.2), but negative effects on the standing load P/2Vs (eqn 2.1).where adaptive divergence (Glossary) along environmental gradients (phenotypic clines (Glossary) for adaptive traits to deviate from their optima . Such migration load would be of concern if gene flow is extensive over LDs (see section ‘How far do seeds and pollen disperse in trees?’) and if phenotypic mismatch of immigrants is mostly due to long-lasting genetic effects  repeatedly suggests that extensive gene flow  has not prevented rapid adaptive divergence of extant populations  have been replicated over a broad range of climatic conditions, provenance (Glossary) reaction norms (Glossary) allow comparison of optimal and original climate for each population: mismatches are not uncommon and often consistent with a maladaptive effect of gene flow at range margins  typically above 0.4 for wood characteristics or phenological traits such as bud set date (stabilising selection (Glossary) is acting to reduce variation within populations (see genetic variance (Glossary) due to gene flow could be substantial  along the typical dispersal distance  is predicted to affect the spatial distribution of genetic variation and diversity of migrants  by helping track the shift of the optimum phenotype through dispersal of pre-adapted genotypes found somewhere else in the range (see eqn 2.2 in response to selection (Glossary) through a general increase in genetic variation (see previous paragraph). Quite generally, dispersal should help adaptation in environments that are changing both in space and time  was only moderately reduced (by 2 days) by the higher pollen and seed dispersal distances of birch along the climatic gradient.Assuming an initially locally adapted population, the new selection pressures induced by climate change will cause the optimal phenotype to deviate from the extant mean phenotype, thus creating a and time . Beyond inbreeding depression (Glossary) is widespread in largely outcrossing species such as trees and inbreeding was found more frequently in isolated marginal populations (heterosis (Glossary) is however not expected generally very strong in trees, due to the combination of large population size and extensive gene flow  in trees  the regional context (e.g. expanding edge or retracting part of the range) and (2) the dispersal syndrome .evolutionary load  and maladaptation increases  with increasing migration  suffice to replenish ronments . We lackbioclimatic envelopes imply that current populations at the trailing and leading edges of the range will face different adaptive challenges. Southern margin populations will face climatic conditions not encountered anywhere in the current species range. Will these challenged populations have the evolutionary potential  masking LDD, (2) dilution effect  and (3) large numbers of putative sources (characterising their positions and genotypes is time- and cost-intensive). Using highly polymorphic genetic markers like microsatellites greatly overcomes the first point, and the advent of next-generation sequencing will improve power and resolution; however, it is still necessary to conceive new experimental designs dealing with points (2) and (3). We propose potential strategies here, mostly relying on a stronger interaction with mechanistic approaches.Experimental dispersal studies monitoring LD pollen and seed dispersal have often been limited in spatial scale due to (1) overlapping of the For wind-mediated gene flow, available weather data could help determine the potential range of pollen and seed dispersal within particular landscapes, regions, or continents. Such an analysis requires regional meteorological datasets, phenological observations over a wide region and sufficient understanding of the meteorological factors driving pollen and seed emission and spread. Products of regional and global weather reanalysis, such as the North American Regional Reanalysis (NARR) dataset and the European Centre for Medium Range Weather Forecast (ECMWF) data, offer useful observational and model-based information on wind, temperature, humidity, radiation and other meteorological data  methods linking pollen, seeds or seedlings to candidate parental populations could be used to evaluate the effective rate and range of contemporary gene flow among discrete populations  may prove efficient in detecting LD gene flow  networks or placed at specific sites in a landscape. Ultimately, dispersal inferences from adequate landscape configurations could enlarge the documented range of dispersal especially for abundant species, in which dilution effects complicate the detection of LDD events in general. Note, however, that the atypical demographic conditions of isolated trees that facilitate LDD assessment may result in observed LDD patterns difficult to generalise, for which modelling approaches may be necessary.An alternative solution, not requiring trap plants but not assessing effective dispersal. The ability of mechanistic approaches to assess dispersal across multiple scales ; although this assumption is unrealistic for many systems in which the dispersal vector moves in a directional manner, such as many seasonal winds, downward flow of rivers, and oriented movement of animals. Similarly, genetic assignment methods for migration rate estimation typically incorporate neither directional nor other kinds of spatial information. Lagrangian dispersal (Glossary) simulations can account for dispersal anisotropy by incorporating turbulence patterns (connectivity maps (Glossary), depicts dispersal probabilities between sites based on large-scale datasets and/or models available for the primary dispersal vector, for example, to assess wind connectivity of plants among islands in the southern oceans , we suggest initiating experiments under controlled conditions mimicking climate change using open top chambers, where the level of CO2, temperature and rainfall can be manipulated. Ideally, such experiments should be conducted for short-generation species  allowing to manipulate as well gene flow among different populations. Selection experiments on short rotation species would not only allow one to monitor genetic changes for key adaptive traits  that are undergoing strong selection under climate change. They would also give access to the rate and direction of change as a function of standing variation and variation due to gene flow, under a wide range of controlled selection regimes.Finally, in addition to experiments conducted in the field  among traits, which may limit or accelerate adaptation to climate change , empirical data are direly missing on patterns of genetic correlations among key ecological traits in trees and on the spatial and temporal variation of their joint selection pressures.The idea that climate change is equivalent to simple spatial shift of local climatic conditions is also a gross simplification. Rather, climatic change may result in new combinations of precipitation patterns, temperature, photoperiod and biotic conditions that occur nowhere within the current range, imposing entirely new selection pressures and favouring the assembly of novel genotypes . Adaptatnge e.g. . ModelliEfforts should be made to fill these gaps and help calibrate models with real data, to ultimately be able to merge evolutionary approaches with niche- and process-based ecological forecasting of climate-induced range shifts.Nemo, Metapop, Trait-based, mechanistic models have recently been developed enabling predictions of species ranges under current and future non-analogous climates e.g.  predictsbioclimatic envelope modelling, variation of gene flow over geographical ranges and evolutionary adaptation of local populations. The basal layer, the climatic layer, defines how changes in climatic conditions over the species’ geographical range modify the localisation of suitable habitats (dispersal kernels (adaptive traits by indicating how far from its local optimum a population might be. Information on gene flow from the second layer will indicate hybridisation rates and fitness effects, depending on the geographical origin of the migrants (A key aspect of the modelling approach advocated here is the overlay of predictions from different processes; ecological niche and habitats . The secmigrants . It willAlthough much emphasis has been placed on the ability of tree populations to migrate fast enough in response to climate change, we have here examined the potential consequences of LD gene flow on their adaptive response to climate change.Many tree species have evolved dispersal syndromes enabling the effective flow of genetic information across distant populations inhabiting contrasting environments. We have argued how such exchanges, although potentially maladaptive in some evolutionary and demographic scenarios, may in the case of forest trees favour adaptation to changing climatic conditions, compensating for their long-generation time. However, we are still missing adequate tools to track LD pollen and seed dispersal enabling to predict population responses to changing environments. The demographic impact of phenotypic mismatch in forest trees also remains to be quantified. Our understanding of the interaction between gene flow and local adaptation under realistic ecological, demographic and dispersal assumptions is still limited, and we have suggested potential theoretical and experimental avenues of research for the integration of dispersal biology, ecology and evolutionary quantitative genetics in a better predictive inferential framework.


Orchomenella franklini . Seven microsatellite loci were used to assess genetic structure on three spatial scales: sites (100 s of metres), locations (1–10 kilometres) and regions (1000 s of kilometres) sampled in East Antarctica at Casey and Davis stations. Considerable genetic diversity was revealed, which varied between the two regions and also between polluted and unpolluted sites. Genetic differentiation among all populations was highly significant  consistent with the brooding mode of development in O. franklini. Hierarchical AMOVA revealed that the majority of the genetic subdivision occurred across the largest geographical scale, with emN≈1 suggesting insufficient gene flow to prevent independent evolution of the two regions, i.e., Casey and Davis are effectively isolated. Isolation by distance was detected at smaller scales and indicates that gene flow in O. franklini occurs primarily through stepping-stone dispersal. Three of the microsatellite loci showed signs of selection, providing evidence that localised adaptation may occur within the Antarctic benthos. These results provide insights into processes of speciation in Antarctic brooders, and will help inform the design of spatial management initiatives recently endorsed for the Antarctic benthos.Currently there is very limited understanding of genetic population structure in the Antarctic benthos. We conducted one of the first studies of microsatellite variation in an Antarctic benthic invertebrate, using the ubiquitous amphipod  Gene flow – or genetically effective migration – is one of the most important factors governing the evolution of species Marine fauna are currently threatened by a plethora of human activities including fisheries harvest, habitat destruction, localised pollution, introduced species and climate change see . InitiatThe Antarctic benthos represents one of the most isolated marine ecosystems on the planet, with particularly unique fauna  genetic subdivision, despite possessing pelagic larvae for dispersal  to address the current paucity of knowledge on intraspecific genetic structure in Antarctic benthos. O. franklini often dominates Antarctic shallow water communities Brooding benthic organisms are particularly interesting candidates in which to address questions of gene flow and speciation in Antarctica, as their lack of a pelagic dispersal phase should lead to high genetic structuring of populations O. franklini. Microsatellites are tandem repeats of typically one to six nucleotides that mutate rapidly and are found at high frequency in most eukaryotic genomes We used microsatellite markers to investigate genetic variation in Orchomenella franklini were collected from two geographical regions, adjacent to the Australian research stations Casey  and Davis , in East Antarctica. Casey and Davis are separated by approximately 1400 km (3) sample of the benthic sediment from no more than 10 m water depth. This sediment was sieved on a 0.5 mm mesh and retained fauna were sorted under a dissecting microscope. All identified O. franklini specimens were removed and stored at 4°C in vials of 80% ethanol  based on a pooled sample of DNA from 15 individual amphipods. SNX forward and reverse linkers were ligated onto size-selected DNA following the procedure of 10, (AAC)10, (GT)13, (CT)13 and (ACAG)7 oligonucleotide repeats by magnetic bead selection A genomic library of O. franklini were used for DNA extractions due to small body size (2–8 mm). DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's instructions, with elution volume decreased to 120 µl to maximise DNA concentration. Microsatellite loci were amplified in three multiplex polymerase chain reactions (PCRs) (Whole specimens of s (PCRs) . For mulOH), unbiased expected heterozygosity (EH) and allelic richness (standardised for sample size: RA) at each locus were calculated in FSTAT 2.9.3 OH, SH and RA in FSTAT. The number of private alleles (AP) at each site was determined using Genalex 6.41 AP, carried out in R 2.12.2 Measures of genetic diversity, including observed heterozygosity (FIS) for each locus-site combination used to determine the nature of those departures (where FIS<0 indicates heterozygote excess and FIS>0 indicates heterozygote deficits). Significance levels were adjusted for multiple comparisons using the sequential Bonferroni procedure Exact tests  were used to test for departures from Hardy-Weinberg Equilibrium, with Wright's fixation index  was used to examine the complete multilocus data set for evidence of distinct genetic groups Cryptic species are common in Antarctic benthic invertebrate fauna FST estimates FST assumes an infinite allele model of mutation (IAM), we also calculated RST which assumes a stepwise mutation model (SMM). Currently there is no consensus over which model is more appropriate for microsatellite data, so the conservative approach is to calculate both FST and RST were calculated in Arlequin 3.11 FST, therefore we also calculated ‘FST and RST) between all sites were generated in Genepop.We calculated Weir and Cockerham's F-statistics FST = 0) at each hierarchical level were determined with 10,000 permutations of the data. We estimated the migration occurring between Casey and Davis as emN = 1/4(1/FST−1) FST among regions generated from hierarchical AMOVA. FST varied considerably among loci (see results), so we tested for evidence of selection at each of the seven loci using Lositan FST’ option The partitioning of genetic variation among regions, locations nested within regions, sites nested within locations and individuals nested within sites was determined using a four-level hierarchical analysis of molecular variance (AMOVA). AMOVA was performed using Hierfstat O. franklini populations within the Casey and Davis regions. We examined the relationship between geographic distance  and genetic differentiation ) between all sites within each region, using Mantel tests implemented in Genepop. The shortest water-based route was an estimation of the direct linear distance (or combination of linear vectors) that an amphipod could feasibly travel between sites . Mantel tests were also performed assuming the SMM (i.e. using RST/(1−RST) for genetic differentiation estimates). Data were permuted 10,000 times to determine significance.Coastal marine populations are unlikely to disperse according to the island model Orchomenella franklini specimens were genotyped for the seven microsatellite loci , compared to Casey , with no evidence of overdispersion. The model revealed strong evidence that AP was significantly greater at Davis than at Casey (p = 0.002), and also significantly greater at unpolluted sites compared to polluted sites , but this made no difference to the significance of genetic differentiation or isolation by distance determined under either the IAM or SSM, therefore results for the raw data alone are presented for simplicity. Evidence of null alleles was also detected at Orcfra5 (in five sites), and Orcfra6 (in three sites). Adjusting allele frequencies at these loci was considered unnecessary because the null alleles were only detected in a small proportion of the total 25 sites.Most populations were in Hardy-Weinberg Equilibrium: of the 175 tests across all loci by all sites, only eight were significant after bonferroni correction . For jus Orcfra4  and all O. franklini from Casey and Davis. Although PCoA explained 67% of the variation in the multilocus genetic data within the first three co-ordinates, it indicated just a single genetic group within the sample  occurring between Casey and Davis was 1.4 .Genetic differentiation among all sites was highly significant, regardless of the mutational model assumed  but not on a smaller scale (i.e. between populations within each of the regions).Locus T values , and we Orcfra12 . We subsOrcfra12  and 4. FWe found evidence of isolation by distance indicative of stepping-stone dispersal among sites within both the Casey and Davis regions. Mantel tests indicated a significant correlation between genetic and geographic distance under both the IAM . There was a clear pattern of isolation by distance within both Casey and Davis, indicating that migration occurs primarily between adjacent populations O. franklini, which predicts highly restricted capacity for dispersal. Indeed, similar findings have been reported for brooding taxa from temperate and tropical regions . Conserving genetic diversity within species is crucial as it provides the raw material for adaptation to changing conditions, hence facilitating long-term persistence The geographical isolation of Clearly, intraspecific genetic structure is a field that warrants increased research in the Antarctic benthos. To date this has been hampered by the logistical difficulties of sampling such an extreme environment Figure S1Results of Principal Coordinate Analysis on multilocus genotypes of Orchomenella franklini. Over 50% of genetic variation is explained within the first two coordinates, however, there is no single group sufficiently discrete to indicate cryptic species in the dataset.(TIF)Click here for additional data file.Table S1Regions, locations, and sites sampled for Orchomenella franklini, with corresponding sample size (n). Locations (and respective sites) in bold have been classified as polluted.(DOC)Click here for additional data file.Table S2Inbreeding coefficients by locus (FIS) and the number of private alleles (AP) for each population of Orchomenella franklini. Asterisks indicate significant departures from Hardy-Weinberg Equilibrium (p<0.05) after Bonferroni correction. Dashes indicate that a locus was monomorphic, hence FIS could not be estimated. Polluted sites are in bold.(DOC)Click here for additional data file.Table S3Matrix of pairwise differentiation estimates for all Orchomenella franklini populations sampled at Casey and Davis.FST below diagonal; RST above diagonal. Estimates of differentiation between Casey and Davis populations are italicised. Negative values have been converted to zero. Polluted sites are in bold.(DOC)Click here for additional data file.


Yersinia pestis infection) is a highly virulent, re-emerging disease, the ecology of which has been scarcely studied in Africa. Human seroprevalence data for the major plague focus of Madagascar suggest that plague spreads heterogeneously across the landscape as a function of the relief. Plague is primarily a disease of rodents. We therefore investigated the relationship between disease distribution and the population genetic structure of the black rat, Rattus rattus, the main reservoir of plague in Madagascar.Landscape may affect the distribution of infectious diseases by influencing the population density and dispersal of hosts and vectors. Plague  in rat populations from four geographic areas differing in topology, each covering about 150–200 kmGenetic diversity estimates suggested that plague epizootics had only a weak impact on rat population sizes. In the highlands of Madagascar, plague dissemination cannot be accounted for solely by the effective dispersal of the reservoir. Human social activities may also be involved in spreading the disease in rat and human populations. Yersinia pestis infection) is a highly virulent disease occurring in many natural ecosystems. One of the major plague foci is Madagascar, where plague has been confined principally to the central highlands since the 1920s. Despite the high prevalence of plague, its ecology has scarcely been studied. Previous studies of plague seroprevalence in humans have suggested that the disease spreads unevenly across landscapes, possibly due to the relief. Plague is primarily a disease of rodents. We therefore investigated the relationship between its distribution and the population genetic structure of its main reservoir, the black rat, Rattus rattus. We used serological and microsatellite-based population genetic analyses to compare four geographic areas of the plague focus . Rat populations had a more marked genetic structure in areas of greater relief. The heterogeneous distribution of plague in the highlands may be related to the population genetic structure of its reservoir, but our results suggest also a role for humans in the dispersion of the disease.Plague ( Yersinia pestis infection) is a highly virulent, re-emerging disease that has caused three pandemics in the last 2000 years Evaluating the contribution of landscape characteristics to spatial heterogeneity in the occurrence of infectious diseases is critical for the assessment of risks and the design of optimal surveillance and control programs. Plague  The genetic structure of flea populations is thought to be strongly determined by host dispersal patterns . In MadaR. rattus?Topographic relief has been shown to influence gene flow in many species , the IPM (Institut Pasteur de Madagascar) and the Madagascan Ministry of Health (Communicable Disease Control Department). Each trapping campaign was validated by the national, regional and local health authorities. Traps were set within houses with the approval of the owner or tenant. Outside houses, traps were set with the agreement of the village head, and always on the edge of cultivated fields, so as not to damage crops. The only rodents sampled were those from introduced species , all of which are considered as pests, with no protected status in Madagascar. No permission was therefore required for their capture.Animals were treated in a humane manner, in accordance with the guidelines of the American Society of Mammalogists Four study areas in which human plague epidemics are regularly reported were compared , Table 1X. cheopis on the rats from houses, S. fonquerniei on the rats from fields) and flea abundance (higher on the rats from houses), and that the movements of rats between habitats are limited Sampling was performed in 1996–1997 (Mandoto), 2000 (Ambositra), 2006–2007 (Betafo) and 2008–2009 (Moramanga), in six to eight villages per area . VillageEach captured rat was euthanized by cervical dislocation, then weighted, measured and autopsied. Blood was collected from the heart on seropads , and a piece of ear or tail was stored in ethanol for molecular analyses.Y. pestis were detected in seropads by a rat IgG anti-F1 enzyme-linked immunosorbent assay  For each sampled rat, specific antibodies against puregenedna purification kit. Genotyping was conducted for 15 microsatellite loci. Six of these loci were originally developed for Rattus norvegicus  within subpopulations and genotypic linkage disequilibrium (LD) between pairs of loci were tested using the Markov chain method implemented in Gicro-checker v.2.2.3 reena (www.montpellier.inra.fr/URLB), to assess the need to correct for null alleles.Some loci displayed significant heterozygote deficiencies in several subpopulations see . In thesS, r, evaluating the number of alleles independent of sample size, calculated for a minimum of 7 individuals by the rarefaction procedure stat v.2.9.3.2 ISF (tested for significance with Fstat). Effective population size (EN) was estimated with Ldner, SH, ISF or EN and plague seroprevalence in each subpopulation within areas by carrying out Spearman's rank non parametric correlation analyses in SAS v. 9.3 r, SH, and ISF between areas with Fstat , and EN using nonparametric Kruskal-Wallis tests in SAS.The genetic diversity of each subpopulation was assessed by calculating Nei's unbiased genetic diversity HS, , allelictructure v.2.3.3 n+1, n being the number of villages sampled in each area). Each run included 50,000 burn-in iterations followed by 500,000 iterations. We also checked that a single mode was obtained in the results of the 10 Structure runs for each K value, by using the Greedy algorithm implemented in Clumpp v.1.1.2 Genetic structure was examined in several ways within the four study areas. Bayesian clustering analyses were first performed with SSTF values stat. We generated 95% confidence intervals (CIs) for the mean STF per area by bootstrap resampling across loci, and we then used Fstat  to compare mean STF. We assessed whether villages may explain population genetic structure within a given area, by performing AMOVA . Independent tests of pairwise genetic differentiation were combined, by the generalized binomial procedure implemented in Multitest v.1-2 We also estimated FST/(1 - FST) against the logarithm of the Euclidean geographic distances between trap sites , excluding intra-village comparisons. The spatial pattern of genetic variation was also investigated by spatial autocorrelation analyses of mean genetic relatedness between pairs of individuals. These analyses complemented standard tests of isolation by distance, as spatial autocorrelation can occur at very fine scales, below the level of the area. Moreover, genetic relatedness provides a more contemporary picture of population genetic structure than the integrative STF. Spatial autocorrelation analyses were performed for each area with Spagedi v.1.2 xyrWe analyzed isolation by distance (IBD) by regressing pairwise estimates of ap sites . Under astat , using pairwise absolute differences between seroprevalence levels and pairwise estimates of STF between subpopulations. A positive correlation would suggest a strong influence of rat dispersal on plague distribution.Finally, we investigated the relationship between plague seroprevalence distribution and genetic structure in rat subpopulations while statistically controlling for the effect of the Euclidean geographic distance. This was done with partial Mantel tests In total, we sampled 1297 rats in the four areas. In the mountain area of Ambositra and the plateau of Moramanga, most of the rats were seronegative, and plague seroprevalence levels were different from zero at only one trap site per area  . In BetaIn total, 910 rats were genotyped. Ten loci  were at HWE. The other five  displayed significant heterozygote deficiencies in several subpopulations, probably due to null alleles. Estimated null allele frequencies were low ISF . In Mandoto, the subpopulation with the highest seroprevalence (MAhiV) also had the highest ISF , unlike effective population size (P = 0.02), with the smallest EN values obtained for Ambositra and the highest ones for Moramanga.The 15 microsatellite loci considered in the analyses were polymorphic in all subpopulations . In AmboFIS  Subpopulmprecise . Spearmahest FIS , but thetructure indicated the presence of a single genetic cluster in Moramanga (minor peak of ΔK not associated with an interpretable structure) and Mandoto (no maximum ΔK), and of two genetic clusters within the mountainous areas of Betafo and Ambositra , with the lowest value obtained for Moramanga and the highest for Ambositra  although most of the genetic variation was observed within subpopulations . There was also significant genetic differentiation between habitats within villages, in each area .Overall mbositra . PairwisP = 0.0001; slope b = 0.009, 95%CI = ) and in Mandoto , but not in Moramanga   (P = 0.09) if the ATitR/AAmpV subpopulations  were excluded.Genetic IBD was significant in Betafo ( 0.007]) . For Amb −0.01]) . HoweverThe analysis of spatial genetic autocorrelation analysis yielded positive values for the first distance classes (within trap sites) in each area. For the second distance class (corresponding to distances between habitats within villages) correlation values were significant and positive only in Betafo and Mandoto. Genetic similarities between individuals subsequently decreased for higher-order classes, becoming mostly non-significant.r2 = 12.0, P = 0.038) or geographic distance . These spurious correlations were related to the subpopulation MAhiV characterized by the highest seroprevalence level of the area . In Betafo, there was no significant correlation between differences in plague seroprevalence and genetic structure  or geographic distance . However, when the subpopulation BAtnV that is also characterized by the highest seroprevalence level of the area, was removed from the analysis, the correlation involving genetic structure became highly significant  . In Mandoto, significant correlations were found between differences in plague seroprevalence levels and genetic structure (the area : when th 0.0001) .R. rattus . Our results were globally consistent with the expectation of an increasing genetic structure of rat populations with topographic relief. Population genetic structure clearly increased from Moramanga (the flattest area) to Ambositra (the most mountainous area). No genetic isolation by distance was detected in Moramanga or Ambositra (STF in EN estimates: STF = 0.10), two subpopulations that are only 3.5 km apart, but on different watersheds. Also, the highly differentiated ATitR subpopulation, despite its central location in the area studied, appears to be very small (EN = 5) and located in an isolated valley. Note however that the linkage disequilibrium method may provide biased downward estimates of EN, in case of pulse migration In all areas, significant but weak genotypic differentiation of mbositra . This maa FST in . In Moratructure results (FST over large distances (Population genetic structure estimates were however intermediate and not significantly different from each other (and from that of Ambositra) in Mandoto and Betafo . Some in results  or pairwistances , indicatistances . This suY. pestis-infected fleas in this area, thereby counteracting the effect of rat population structure on disease spread . Alternatively, rat population structure may play a less important role than human social interactions in determining the distribution of plague seroprevalence in rats and humans of this area. High levels of human movements in Mandoto may favor the translocation of Y. pestis-infected rodents and/or fleas between villages, thereby spreading plague in both the human and rat populations independently of their genetic or geographic proximity. There is ample evidence for the influence of human traffic on the spread of plague over larger distances, both in Madagascar and elsewhere Y. pestis transfers may also be important at a more local scale.Population genetic analyses indicated similar effective dispersal rates of rats in Betafo and Mandoto. Nevertheless, plague seroprevalence distribution was related to genetic structure in Betafo, but not in Mandoto . The rolIn the mountainous area of Betafo, the distribution of seroprevalence levels was clearly related to genetic structure when pairwise comparisons involving BAtnV are removed . The posIFS tended to be higher in subpopulations with positive plague seroprevalence in each area, especially in MAhiV and BAtnV , together with longitudinal surveys of plague seroprevalence in human and rat populations.


Petroica traversi) which, in 1980, was reduced to a single breeding pair. Following this bottleneck, some females were observed to lay eggs on the rims of their nests. Rim eggs left in place always failed to hatch. To expedite population recovery, rim eggs were repositioned inside nests, yielding viable hatchlings. Repositioning resulted in rapid growth of the black robin population, but by 1989 over 50% of all females were laying rim eggs. We used an exceptional, species-wide pedigree to consider both recessive and dominant models of inheritance over all plausible founder genotype combinations at a biallelic and possibly sex-linked locus. The pattern of rim laying is best fitted as an autosomal dominant Mendelian trait. Using a phenotype permutation test we could also reject the null hypothesis of non-heritability for this trait in favour of our best-fitting model of heritability. Data collected after intervention ceased shows that the frequency of rim laying has strongly declined, and that this trait is maladaptive. This episode yields an important lesson for conservation biology: fixation of maladaptive traits could render small threatened populations completely dependent on humans for reproduction, irreversibly compromising the long term viability of populations humanity seeks to conserve.Conservation management often focuses on counteracting the adverse effects of human activities on threatened populations. However, conservation measures may unintentionally relax selection by allowing the ‘survival of the not-so-fit’, increasing the risk of fixation of maladaptive traits. Here, we report such a case in the critically-endangered Chatham Island black robin ( Geospiza fortis) at Academy Bay in the Galápagos Islands Humans impose considerable selection pressures on wildlife populations worldwide Petroica traversi), a critically endangered passerine  was observed to lay rim eggs  . During If rim-laying has a genetic basis, and its spread has been facilitated by human intervention through egg repositioning, the frequency of this trait would be predicted to decrease following cessation of intervention. Repositioning of rim eggs ceased in 1990; we therefore compared egg-laying data from three years before cessation of repositioning (1987–89) with a three year period almost two decades after management stopped (2007–09). Consistent with relaxed selection during repositioning of rim eggs, the occurrence of rim eggs has decreased significantly between these two periods  . To ensuWe next assessed whether rim-laying behavior has an observable fitness effect, using black robin breeding data from 2007–11. When rim eggs were not repositioned, females that laid rim eggs had significantly reduced clutch sizes (i.e. number of eggs laid inside nests that were incubated), and decreased hatching and breeding success compared to normal-laying females, demonstrating that rim laying substantially reduces fitness .The human-assisted spread (between 1984–89) and subsequent decline of rim laying following cessation of intervention (2007–11) are consistent with, but do not demonstrate a genetic basis for this trait.To examine whether rim laying is consistent with a genetic explanation, we evaluated pedigree data from the entire black robin population from 1980–89 . A tempokelihood  since alTo gain insights into the yearly increase of the dominant allele A under Model 4 during the managed phase between 1980 and 1989, we inferred the trajectories of the number of A alleles from the compatible genotypic configurations that are readily available from our SMC algorithm over the entire population pedigree. The mean and confidence sets of the trajectories of allele A as well as the total number of alleles (twice the population size) are given in We next sought to rule out the possibility that rim-laying is an individual response to environmental conditions. Traditional segregation analyses We have documented the emergence, and human-assisted spread of a maladaptive egg-laying behavior with a genetic basis in the endangered Chatham Island black robin. Human intervention, by relaxing selection against rim-laying individuals, facilitated a rapid increase in the frequency of this deleterious dominant trait. We surmise that the spread was facilitated by the egg repositioning that rendered this otherwise maladaptive trait effectively neutral, enabling its spread via strong genetic drift and high levels of inbreeding within an extremely small population. The human-assisted spread of this maladaptive trait highlights a dilemma that conservation efforts face worldwide: rapid population increase is crucial to avert extinction of severely threatened species, but management practices that reduce mortality and increase fecundity may inadvertently relax selection, which may in turn lead to the fixation of deleterious alleles.Inadvertent fixation of deleterious alleles risks undermining the ultimate goal of conservation: the reestablishment of viable, self-sustaining populations in the wild. Hence, conservation planning has to overcome this fundamental dilemma of rapidly increasing the size of severely endangered populations to avoid immediate extinction but without simultaneously increasing the frequency of detrimental alleles that are already present in the population and risking their potential fixation.Bombyx mori) provides the most stunning example of this. Over the course of 5,000 years it has become utterly dependent on humans for reproduction and survival Relaxation of selection following from human activity is a lesson well known from domestication, where artificial selection has made some domesticated species entirely dependent on humans for successful reproduction In contrast, our results show that the black robin just narrowly escaped such a fate. During the initial management period, when eggs were repositioned, the rim-laying trait rose in frequency to over 50% of females. Conditions favoring relaxed selection on the rim-laying trait have, to our knowledge, been absent for two decades, and selection against rim-laying has significantly decreased the frequency of this trait . HoweverAll research was carried out with appropriate permits from the Animal Ethics Committee at the University of Canterbury and the Department of Conservation in New Zealand.Petroica macrocephala chathamensis) nests to ensure that they were incubated. From 2007–11, nests were monitored to record the occurrence of rim eggs, but rim eggs were not repositioned.Chatham Island black robin populations were monitored between 1980–89 on Mangere and Rangatira Islands , and between 2007–11 on Rangatira Island. Birds were color-banded to allow individual recognition. Nesting attempts were monitored, recording clutch size, rim eggs and parentage of offspring. From 1980 to 1989, eggs laid on the rim were repositioned inside nests or relocated to Chatham Island tomtit  fit by the Laplace approximation (lme4 package in R v2.12.2) To test whether rim laying leads to a substantial fitness loss in black robins, we again used GLMMs as described above with binomial or Poisson errors to compare effective clutch size, hatching success and breeding success of normally-laid clutches and those that included rim eggs. Female identity was included as a random effect in all analyses and LRTs were performed to obtain a more conservative measure of the effect of a predictor variable on the response variable. Parameter estimates (±1 se) of all five GLMMs are given in We use a sequential Monte Carlo (SMC) Based on this likelihood as a test statistic we develop an exact phenotype permutation test for non-genetic versus genetic factors. The phenotype permutation test is described in detail in Table S1Parameter estimates (±1 se), z statistics and P-values of generalized linear mixed models testing whether occurrence of rim eggs varied over time  and whether rim laying incurs fitness costs (C–E). In all models female identity was included as a random effect. P-values presented here are not the same than those presented in the text. In the text, P-values of the more conservative likelihood ratio tests are presented.(DOC)Click here for additional data file.Appendix S1Pedigrees, likelihoods and tests.(PDF)Click here for additional data file.


Ophiognomonia clavigignenti-juglandacearum (Oc-j), the butternut canker fungus, has caused range-wide mortality of butternut trees in North America since its first observation in 1967, the history of its emergence and spread across the United States and Canada remains unresolved. Using 17 single nucleotide polymorphic loci, we investigated the genetic population structure of 101 isolates of Oc-j from across North America. Clustering analysis revealed that the Oc-j population in North America is made up of three differentiated genetic clusters of isolates, and these genetic clusters were found to have a strong clonal structure. These results, in combination with the geographic distribution of the populations, suggest that Oc-j was introduced or has emerged in North America on more than one occasion, and these clonal lineages have since proliferated across much of the range of butternut. No evidence of genetic recombination was observed in the linkage analysis, and conservation of the distinct genetic clusters in regions where isolates from two or more genetic clusters are present, would indicate a very minimal or non-existent role of sexual recombination in populations of Oc-j in North America.The occurrence of multiple introduction events, or sudden emergence from a host jump, of forest pathogens may be an important factor in successful establishment in a novel environment or on a new host; however, few studies have focused on the introduction and emergence of fungal pathogens in forest ecosystems. While  Cryphonectria parasistica, that nearly eliminated all mature American chestnut trees, which was once a dominant hardwood species in the eastern United States. In addition to chestnut blight, several other important exotic fungal diseases have greatly affected the North American forest landscape of the last century. These include, but are not limited to, Ophiostoma novo-ulmi causing Dutch elm disease of American elm, Ophiognomonia clavigigenenti-juglandacearum causing butternut canker on butternut, Cronartium ribicola causing white pine blister rust on white pine, Phytophthora ramorum causing sudden oak death on coastal live oak, Discula destructiva causing dogwood anthracnose on flowering dogwood, and most recently, Geosmithia morbida causing thousand cankers disease, which is damaging black walnuts in the western United States and poses a substantial threat to black walnut plantations throughout North America.Invasive species continue to be a major threat to North American forests, and pose an important threat to forest biodiversity and the ecosystem services they provide. Much of the attention related to invasive species has focused on human and animal disease; however, invasive pathogens of plants have the capacity to greatly affect humankind through the devastation of agricultural crops and the destruction of forest ecosystems. It is estimated that invasive plant pathogens cause $21 billion in crop losses each year in the United States, and $2.1 billion of forest products are lost each year to alien pathogens , was described for the first time  clavigignenti-juglandacearum as an endophyte of Acer truncatum .In many cases involving invasive forest pathogens, there can be an extended latent period between infection and symptom development, and this delay often results in diseased trees being overlooked. It is therefore quite difficult to determine if the new pathogen was introduced, if subsequent introductions may have occurred, and how far the pathogen has spread since its original introduction. In the case when a source population cannot be identified, it is also possible that the emergent pathogen was derived from an avirulent strain, such as an endopyhyte, on the same host or has made a host jump from a plant species that was either introduced into the new host's range or has migrated into the new host's range due to climate change. A good example of this situation is the case of the butternut canker pathogen, um Oc-J; , which hum Oc-J; , and in Oc-j has recently emerged as a pathogen of butternut in North America  What is the relative importance of outcrossing and clonal reproduction in Oc-j populations? (ii) Is there evidence of multiple introduction or emergence events? (iii) Are populations of Oc-j geographically  or genetically structured?The aim of this study was to investigate the genetic diversity of Oc-j were collected from diseased plant tissue from butternut (Juglans cinerea), heartnut, (J. ailantifolia var. cordiformis), and black walnut (J. nigra) from locations throughout Ontario in 2009 and 2010 . DNA purity and quantity were estimated using a Nanodrop photospectrometer.Isolates of 2, 1 μL containing 10 mmol/L each dNTP, 0.25 μL of GoTaq Taq polymerase, 5 μL each of 5-μmol/L concentration of forward and reverse primers, 2 μL of DNA at a concentration of 10 ng/μL, and 21.75 μL of sterile deionized water. PCR parameters were 95°C for 5 min; followed by 35 cycles of 95°C for 1 min, 54°C for 1 min, 72°C for 1 min; and completed with 72°C for 5 min followed by 4°C. PCR products were purified using Qiaquick spin columns . For sequencing, 2 μL of 5 pmoles/μL primer was added to 2 μL of purified PCR product (20 ng/μL). Amplified products were sequenced with the BigDye version 3.1 ready reaction kit  on an ABI 3730 automated sequencer at the University of Guelph Genomics Facility or the Hubbard Center for Genome Studies at the University of New Hampshire. Sequencing chromatograms were visualized, and sequences were aligned and edited using BioEdit , taking into account departure from Hardy–Weinberg equilibrium, was estimated for the sample set using GenClone 1.0  method  in MEGA version 4.0. All positions were included in the analysis and relative support for the branches was estimated with 1000 bootstrap replications. In addition, a set of 95% plausible haplotype networks, for the Oc-j is not well understood, it is possible that the fungus emerged from an alternate host and/or was introduced on more than one occasion. Therefore, the genetic structure of the population may not coincide with geographic proximity of individuals, but rather are clustered based on their genetic relatedness. To test the geographic and genetic structure of populations, we used three methods. The first was a series of nonparametric tests applied in a hierarchical manner by the program SNAP Map  empirically determined by comparing posterior distribution likelihoods among runs. Although Structure has been used to study genetic relationships among races, cryptic species or to detect regional substructures in fungal pathogen species, violation of hypotheses such as linkage disequilibrium in clonal subgroups can sometimes lead to incorrect assignments. We therefore, also used a principal component analysis (PCA) to investigate the genetic structure of the Oc-j population in North America. As PCA is independent of any genetic hypotheses, such as Hardy–Weinberg equilibrium, it is suitable for the analysis of clonal or partially clonal species. PCA analysis was performed using the Adegenet package under the R-software. For both the Bayesian and PCA analyses, only the clone-corrected data set was used to give identical weight to each multilocus genotype.The analysis with Structure can be used to estimate parameters independently of the posterior probability distribution of allele frequencies. Parameters are estimated under the null model of panmixia, where each locus is at Hardy-Weinberg equilibrium and independent of the others. Using the admixture model, we estimated the number of genetic clusters, between o et al.  was usedR), the Simpson evenness index (V), and the complement of the slope of the Pareto distribution of clonal membership in the program GenClone. These measures are recommended as the most parsimonious set of non-redundant indices of clonal diversity  of the haplotypes were found to have multiple isolates with three haplotypes  having eight or more isolates making up 32% of the isolates sampled. Haplotype H1 had the most isolates (15) and included isolates from the NE, NC, and ONT populations. H16 had the second largest number of isolates (9) and also included isolates from the NE, NC, and ONT populations. In contrast, all eight isolates of H44 were recovered from Ontario. All neutrality tests were non-significant, indicating these polymorphic loci are selectively neutral. Tests for linkage disequilibrium among the five regions found significant LD overall , and in pairwise tests of the five genomic regions, significant LD was also detected ([IA] = 0.62 P = 0.023).Based on the combination of 17 polymorphic loci within non-coding regions of the Snn compared isolates from the six populations in the U.S. and Canada. There was no significant difference between the populations from the northeast and southeast (P = 0.12), Wisconsin, Minnesota, and Indiana (P = 0.34), and Ontario remained its own group as there was no significant difference (P = 0.23) between the south-central and eastern Ontario populations . The result was a putative North American population of three groups designated northeast/southeast (NE/SE), northcentral (NC), and Ontario (ON)  .q> 0.75 for assignment to a particular cluster, 84% of the isolates belonged to only one cluster, indicating that the three clusters were highly differentiated . Taken together, these analyses point to a strongly clonal reproduction regime for each of the genetically distinct clusters, using both the complete and clone-corrected datasets.All genetic clusters had a clonal structure, as shown by the number of repeated haplotypes, or haplotypes that were only distant by 1 allele . The SimThe frequency of isolates from each of the genetic clusters among regions was calculated using all isolates. Isolates belonging to cluster 1 were found in all four regions of North America and were the most abundant genetic cluster in each region . IsolateOc-j. While numerous reports and papers have been published on the progression of the butternut canker epidemic since its first appearance in the 1960s  commonly known as heartnut, which is a close relative of butternut and has been known to hybridize with butternut. While Oc-j does not kill Japanese walnut, it has been isolated from small branch cankers on the tree. The one isolate from J. ailantifolia in this study was found to belong to haplotype H1, the most frequently recovered haplotype, which also appears to be ancestral to many of the other haplotypes. Further isolation and genotyping of Oc-j from J. ailantifolia will help determine the role of this tree in the introduction and spread of Oc-j.The low level of nucleotide variation and the high proportion of low-frequency haplotypes detected in the isolates . This poanalyses , 6, Bayeanalyses , 4 and sanalyses  or infecanalyses . In addiOc-j into different regions of North America may explain the geographic population subdivision identified using Hudson's Snn test and verified in the AMOVA pairwise comparison of subdivided populations.A third potential scenario is the introduction of the fungus on wood or lumber products imported into the United States. Major ports of entry for wood products into the eastern North America include several locations along the east coast as well as through the St. Lawrence Seaway, which officially opened in 1959, marking the first arrival of an oceangoing ship in the Duluth-Superior harbor in Minnesota. While the St. Lawrence Seaway has been credited with the introduction of many aquatic invasive plant species into the Great Lakes ecosystem . In addition to causing the distinctive trunk cankers that lead to the mortality of butternut trees, Oc-j has been recovered from young stems, terminal buds, leaves, flowers, and seed pericarps and cotyledon on butternut, as well as branches and seed pericarps of black walnut and branches and flowers of heartnut  as southern populations began to recolonize areas previously covered by glaciers (Hoban et al.  cluster . FurtherOc-j populations cluster into three genetic groups provide new insight into the population genetic diversity and evolution of Oc-j, and support a model of multiple recent introduction or emergence events of Oc-j clonal lineages in North America. These findings also provide an example of how multiple introduction or emergence events are likely required for a pathogen to become resident in a new environment. In addition, once a new pathogen has established in a location on a novel host, there is still potential for a more virulent strain of the pathogen to emerge. Therefore, quarantine procedures may need to take into consideration not just the fact that the invasive pathogen has become established, but the likelihood of a more virulent strain being introduced and causing greater mortality than has already occurred. However, further genotyping studies are needed to provide a more complete picture of the host range, geographic range, and interactions among the clonal lineages. Additional pathogenicity experiments are also planned to evaluate the roles isolate virulence and host genotypes may play in the population structure of Oc-j and the dominance of specific clonal lineages in different regions of North America.In summary, our findings that North American