Senecio inaequidens (Asteraceae) in southern France, where it was introduced at a single wool-processing site. It gradually invaded the Mediterranean coast and the Pyrenean Mountains, which have noticeably different climates. We used seeds from Pyrenean and Mediterranean populations, as well as populations from the first introduction area, to explore the phenotypic variation related to climatic variation. A reciprocal sowing experiment was performed with gardens under Mediterranean and Pyrenean climates. We analyzed climatic phenotypic variation in germination, growth, reproduction, leaf physiology and survival. Genetic structure in the studied invasion area was characterized using AFLP. We found consistent genetic differentiation in growth traits but no home-site advantage, so weak support for LA to climate. In contrast, genetic differentiation showed a relationship with colonization history. PP in response to climate was observed for most traits, and it played an important role in leaf trait variation. EME mediated by seed mass influenced all but leaf traits in a Pyrenean climate. Heavier, earlier-germinating seeds produced larger individuals that produced more flower heads throughout the growing season. However, in the Mediterranean garden, seed mass only influenced the germination rate. The results show that phenotypic variation in response to climate depends on various ecological and evolutionary processes associated with geographical zone and life history traits. Seeing the relative importance of EME and GD, we argue that a “local adaptation vs. phenotypic plasticity” approach is therefore not sufficient to fully understand what shapes phenotypic variation and genetic architecture of invasive populations.When exotic species spread over novel environments, their phenotype will depend on a combination of different processes, including phenotypic plasticity (PP), local adaptation (LA), environmental maternal effects (EME) and genetic drift (GD). Few attempts have been made to simultaneously address the importance of those processes in plant invasion. The present study uses the well-documented invasion history of  Climate, for instance, often varies gradually over the invasion range at both regional and continental scales. Clinal variation in life history traits , associated with climate, have been documented in several invasive plants Within a species distribution, climatic variability generates heterogeneous selection pressures. Local adaptation (LA) can be defined as the result of selection pressures exerted by local environmental conditions on a population's genetic pool. Genetic differentiation of invasive plant populations can be the result of selection combined with restricted gene flow The genetic structure of alien species in the invaded range is not only the result of selection. Genetic drift (GD), i.e. the random fluctuation of allele frequencies, can also shape the genetic structure vs non-fitness traits and proposed that a successful invader can benefit from plasticity as i) a Jack-of-all-trades, able to maintain fitness across a range of environmental conditions, notably through the plasticity in morphological and physiological traits and/or ii) a Master-of-some, better able to increase fitness in favorable environmental conditions.Phenotypic plasticity (PP) is the change in phenotypic expression of a genotype in response to environmental conditions In the literature, PP and LA have often been studied as two potential mechanisms for the success of invaders  can also modulate phenotypic variation in plant populations and mediate population success across generations. In a broad sense, EME are maternal phenotype influences on offspring phenotype that occur via mechanisms exclusive of any genetic information Senecio inaequidens (Asteraceae) These different sources of phenotypic variation, associated with climate, have been studied in invasive plant species Reciprocal common gardens are useful tools to examine the mechanisms responsible for the phenotypic variation within plants In the present study, we explored phenotypic variation in response to climate and evaluated the extent of LA, GD, PP and EME in germination, growth, ecophysiological and reproductive traits at the population level. The first step of the present study was a reciprocal sowing experiment. An experimental garden was placed in each of the two most extreme climatic zones colonized by a rapidly invasive plant species after migrating from a single introduction site. We subsequently conducted a quantitative analysis of phenotypic variation in life history traits. The second step was a quantitative assessment of the species genetic structure using Amplified Fragment Length Polymorphism (AFLP) markers in order to better understand the patterns of genetic differentiation during invasion.Senecio inaequidens DC (Asteraceae) is considered a particularly suitable model for evolutionary studies The model species S. inaequidens is a herbaceous perennial native to Southern Africa, where it occurs as two co-existing cytotypes. Only tetraploids are reported in Europe Ex situ germination tests have indicated that germination is absent at temperatures below 2°C th century, recorded independent introduction sites chronicle the species migration routes As a result of wool importation from South Africa to Europe during the late 19S. inaequidens towards distinct climatic areas makes it an ideal model to study the response of invasive plant populations to climatic variation. A previous study indicated population differentiation along a climatic gradient in southwestern France The species is not extensively propagated for ornamental purposes, thus reducing the risk of anthropogenic gene flow among regions. The particularly well-documented gradual expansion of Armissan, Narbonne and Narbonne-Plage); 2) the original introduction area, i.e. in the vicinity of the wool-processing industries of Mazamet ; and 3) high plateaus in the Pyrenean region  (see Three populations were selected in each of the following climatic zones: 1) the Mediterranean coast (one) see . The throne) see . The oriMediterranean garden; alt.: 55 m; 43°38′N; 3°52′E), and the other under the Pyrenean mountain climate . Two identical random block common gardens were established in two of the climatic zones. One was representative of the Mediterranean climate . Each of the two lots was randomly assigned to a garden, with offspring from a field-collected plant representing a set of half or full siblings. Gardens were composed of 15 blocks of nine pots, and were surrounded by an additional row of potted plants to prevent edge effects. The pots were placed 80 cm apart to prevent competition and contained 2.5 l sand, 2.5 l compost and 0.5 l hydro-granulates. We used pots filled with a uniform substrate in order to eliminate environmental variation other than climate. On March 20th (Mediterranean garden) and March 21st, 2008 (Pyrenean garden), the 10 seeds per lot were collectively sown in the pots. Pots were saturated with water and covered with protective light-permeable canvas  until germination was complete to prevent seed predation and/or wind dispersal. Seedlings were counted every 2–3 days. The first emerged seedling in each pot was marked for measurement . Other seedlings were removed after counting. The time to germination was recorded, and represented the number of days between sowing and the emergence of the first seedling. The germination rate (%) was defined as the proportion of germinated seeds among the ten sown in each pot. During each month of the flowering period, all ripe flower heads were counted and clipped. For the last measurement, flowering capitulae and buds were counted as well as ripe flower heads. Flower head production was the sum of all capitulae over the growing season , and was used as a proxy for fitness. In mid-October 2008, maximum height (h) and two perpendicular diameters (d1 and d2) were measured for all (surviving) plants. Plant volume (dm3), another fitness-related trait, was calculated as 2/48plant volume = π*h*(d1+d2) (volume of a cone). From September 22nd to 26th, six leaves per plant were randomly collected in 10 of the 15 blocks in each garden. Selected leaves were fully expanded and did not show herbivore damage. Individual mean specific leaf area  and leaf dry matter content  were estimated according to Garnier et al. We randomly selected two lots of 10 seeds from each collected capitulum. Seeds without obvious anomalies were chosen and weighed  to the nearest 0.1 mg  for the reciprocal sowing experiment.Since field populations were not on privately-owned or protected locations and no endangered or protected species were involved, specific permits were not required for seed collection. All necessary permits were obtained from the French The mass of field-collected seeds was analyzed by applying a two-way ANOVA with climatic zone  as the fixed factor and population (nested within climatic zone) as the random factor. In the case of a significant climatic zone effect, Tukey's multiple comparisons test was used.Phenotypic traits in the reciprocal sowing experiment were analyzed. First, a four-way ANCOVA model was run with garden (G) and climatic zone (Z) as the fixed factors, population  and block  as the random factors and seed mass (SM) as the covariate. All possible interactions between factors were included in the model. The block factor and interactions were not significant, and were therefore removed from the model to increase the statistical power. A three-way ANCOVA model was subsequently used to analyze the effects of the interactions between the covariate and the climatic zone, garden and population factors. The interactions between factors and the covariate were then removed from the model to analyze the effects of individual factors, the covariate and interactions between factors. A correct test cannot be made for factors when covariate×factor interactions are included in the ANCOVA model, because the null hypothesis is an equal ordinate at the origin Each factor, covariate or interaction represents a source of phenotypic variation in relation to climate that potentially explains the variance in phenotypic traits. The garden factor reflects PP induced by climate at the population level. The climatic zone factor reflects genetic differentiation between distinct invaded regions, potentially due to LA if local populations outperform distant localities at the home site. If this is not observed, differences between zones are attributable to GD and colonization history. It has to be noted that LA can only be evidenced for populations from the Pyrenean and Mediterranean zones, since there was no garden in the original introduction area.The random factor population corresponds to genetic differentiation within climatic zones. A significant garden×climatic zone interaction or garden×population interaction demonstrates genetic differentiation among and within climatic zones, respectively, on the level of PP in response to climate. The seed mass covariate suggests a role of EME on phenotypic variation. A significant seed mass influence indicates that seed provisioning by maternal plants affects offspring life history traits. A significant interaction with the garden factor demonstrates that the expression of EME is dependent on climate, whereas a significant interaction with the climatic zone or population factors indicates genetic control of EME. No transformation was necessary to meet the assumptions of statistical analyses. Analyses were performed with Minitab ver. 15.1.30 . In order to analyze the effects of seed mass, garden, climatic zone, population and the interactions garden×climatic zone and garden×population on survival, we fitted a generalized linear model  to our data with a binomial error distribution and a logit link function. The predictors were added sequentially. Chi-square statistics were used to calculate the predictor significance.EcoRI and 2 units of MseI  and ligated to the appropriate double-strand adaptor  using 1 unit of T4-DNA ligase  in a total volume of 40 µl. Following two hours of incubation at 37°C, preselective amplification was performed with EcoRI-A and MseI-C preamplification primers  from 3 µl of the digested-ligated DNA mixture (10× diluted). Amplifications were performed with a PTC 200  as follows: 2 min at 72°C; followed by 25 cycles of 30 s at 94°C, 30 s at 56°C, 2 min at 72°C; and a final extension step for 10 min at 72°C. Samples were cooled to 4°C. Preselective DNA amplification products were diluted 20×, and 5 µl were used in a selective PCR amplification. EcoRI primers were labeled with the 6-FAM fluorescent dye set. Selective PCR parameters were as follows: 10 min at 94°C; followed by 13 cycles of 30 s at 94°C, 1 min at 65–55.9°C (delta T = −0.7°C/cycle), 1 min at 72°C; followed by 23 cycles of 30 s at 94°C, 1 min at 56°C, 1 min at 72°C; and a final extension step for 10 min at 72°C. Samples were cooled to 4°C. A total of two primer pair combinations  were selected for twelve primer combinations on the basis of the quality and quantity of generated bands. Two repetitions of AFLP reactions were performed  on twenty individuals to test the repeatability of reactions. A blank control (i.e. no template) was included to detect the presence of contamination. Genotyping was conducted on an ABI PRISM 3100, using a pooled mix of 1.5 µl PCR product, 10 µl deionized formamide, and 0.2 µl GS500ROX size standard . AFLP patterns were visualized with GeneMapper software ver. 3.7 . Highly repeatable and reliably detectable loci were identified using duplicate samples and manual inspection before automatic scoring for the presence/absence (1/0) of selected bands in each individual. Bands of identical size amplified with the same primer were considered homologous.Seeds from 20 individuals from each population were sown under greenhouse conditions and one descendant per sampled individual was maintained. Descendants  were sampled for genetic analysis. Genomic DNA was extracted from 100 mg of fresh leaves using the DNeasy™ extraction kit . AFLP was performed as previously described a priori geographic groupings was explored using the Bayesian clustering algorithm implemented in STRUCTURE ver. 2.1 www.viamichelin.fr. Road distance is potentially more relevant than geographic distance because S. inaequidens is known to follow roads and railways during colonization in situ history, while a loss can be interpreted as a sign of GD. DW statistics were compared between climatic zones, based on population values using Mann-Whitney U tests.Genetic structure was assessed by applying several approaches. The presence of differentiated gene pools in the overall sample without 2,6 = 9.26; P = 0.015) and between populations within zones . Mean ± SE were 37.6±0.56 10−4 g in the Pyrenean zone; 31.8±0.56 10−4 g in the introduction zone and 32.5±0.46 10−4 g in the Mediterranean zone. Tukey simultaneous tests indicated that seeds from the Pyrenean zone were significantly heavier than seeds from the introduction zone  and the Mediterranean zone . However, seed mass was not significantly different in the latter two zones .Seed mass (i.e. mass of lots of 10 seeds) significantly differed between climatic zones , no variation in time to germination was recorded in the Mediterranean garden. However, in the Pyrenean garden, the germination window reached 60 days. The analysis of different sources of variation in phenotypic traits is presented in The Mediterranean and Pyrenean gardens showed germination in 135 and 130 pots, respectively, where two and twelve plants did not bloom, and five and eight plants died before plant volume was measured. As germination in all pots occurred between two rounds of observation . Exclusive of germination rate, the influence of seed mass was strongly conditioned by climate, indicated by a high garden×seed mass interaction. Seed mass strongly affected germination rate and timing, growth and reproductive output under the Pyrenean climate. However, it only influenced germination rate under the milder Mediterranean climate .P<0.001), with 57 and 13 plants surviving in the Mediterranean and Pyrenean gardens, respectively. The predictor population was also significant . Other predictors did not affect survival .Winter survival differed according to the garden . Bayesian analysis in STRUCTURE was applied for the overall analysis of individuals, which yielded the best clustering of data for K = 1, ((LnP(D|K = 1)) = −6401), suggesting the sample is comprised of a single genetic unit.A total of 106 scorable fragments were obtained, with 94% polymorphism observed. Differentiation among populations was low but significant , 2.9% to differentiation among populations within climatic zones (P<0.001) and 96.1% to within-population variation. The Pyrenean zone was primarily responsible for climatic zone differences, exhibiting significant Fst associated with the Mediterranean zone  and the introduction zone . The pattern of genetic structure also showed high genetic differentiation between populations within climatic zones, with the exception of the Mediterranean zone. Pairwise genetic differentiation (Fst) between populations ranged from 0 to 0.051. Fst values were significant (P < 0.001) between the La Llagone population and all other populations (mean ± SD  =  0.039±0.007), and significant (P<0.05) between Enveitg and Mazamet; Egat and Armissan; and between Castaunouze and Egat, Hautpoul and Narbonne. This pattern was confirmed by the neighbor-joining unrooted dendrogram . The species level global genetic diversity (HT) was 0.2964 (SE  =  0.004814). Genetic diversity (H) of individual populations ranged from 0.267 (Castaunouze) to 0.316 (La Llagone) (mean ± SE  =  0.2917±0.002143) (Enveitg) to 16.2 (Narbonne-Plage) (mean ± SE  =  14.1±1.31). Pyrenean populations (mean DW  =  12.89) presented globally lower DW values than other populations .The pairwise Fst values among the nine populations were correlated with pairwise geographic and road distances  . Differe.002143) . DW statS. inaequidens. Our approach voluntarily eliminated phenotypic variation related to other (e.g. edaphic or biotic) environmental factors to better examine the role primary sources of phenotypic variation play in the rapid and successful invasion of an introduced species in distinct climates. Our analysis of LA, PP, EME and GD therefore only corresponds to how the species responded to climatic variation during its invasion in southern France. To encompass climatic variation and genetic variability in this invaded range, we considered populations from three distinct climatic zones: the original introduction area (Mazamet) and two areas situated in the most extreme climates in this region, the high Pyrenean mountains and the Mediterranean coast. Since we could not set up an experimental garden in Mazamet, our analysis of LA, PP, EME and GD is exhaustive only for populations from the Pyrenean and Mediterranean regions. Populations from the original introduction area must essentially be seen as representative of an intermediary climate to understand the patterns of variation in the considered invaded range.In the present study, we analyzed the phenotypic variation induced by climatic variation within the invaded range of The results of the reciprocal sowing experiment and genetic analyses revealed that phenotypic variation in relation to climate involved more sources of phenotypic variation than previously reported in this species. Clinal differentiation in growth traits, as previously observed S. inaequidens. However, over the growing season, reproductive output did not differ between gardens. Therefore, S. inaequidens appears capable of successful reproduction throughout its invasion range in southern France, despite different plastic responses. This corresponds to the Jack-of-all-trades situation described by Richards et al. data not shown). SLA and LDMC reflect a fundamental trade-off in plant function between a rapid production of biomass  and increased drought resistance . These traits are well-known to respond to environmental conditions PP is more directly measured by comparing phenotypes of the same genotype in different environments. However, estimates of PP at the population level allow for a more realistic evaluation of its ecological role in natural populations The significant interaction between climatic zone and garden indicated that plants of Pyrenean origin were less plastic in SLA than others. This suggests that lower PP did not counteract the colonization of remote areas.Similar reproductive output in both gardens indicated similar average one-year fitness in both climates. However, due to increased mortality following the harsher Pyrenean winter, longer-term fitness should be higher in the Mediterranean, enhancing future invasion.S. inaequidens are interpreted as a sign of LA S. inaequidens under the two climates could instead be the result of genetic differentiation in allocation pattern, with plants originating from the Mediterranean zone allocating more resources to growth than reproduction and Pyrenean plants allocating more resources to reproduction, e.g. through increased seed mass.Since plant size can be considered a proxy for fitness S. inaequidens. Further research is needed to fully understand the clinal trend in plant size in this species, but the present study suggests climate does not act as a major selection pressure.Since our approach specifically focused on phenotypic variation in response to climate, the lack of evidence for LA in the present study does not exclude that populations may have adapted to other environmental parameters. A recent study S. inaequidens population age, and several isolated populations are bottlenecked across Europe. Our AFLP analysis also showed evidence for high within-population genetic diversity, which is expected in an outcrossing tetraploid species.In contrast, evidence for neutral genetic differentiation was detected within the invaded area in southern France. Despite a limited number of loci, the AFLP analysis helped to better understand the role of GD and colonization history in the genetic structure of the species. Differentiation was low, indicating significant gene flow within each location, which is consistent with the invasive history of this species in France based on the literature. STRUCTURE analysis confirmed that all sampled populations originated from the introduction area of Mazamet, and It has to be noted that if the genetic differentiation observed is mainly driven by genetic drift and colonization history, it is not expected to explain invasion success in contrasted climatic environments.Seed mass was an important source of phenotypic variation in the Pyrenean garden. In contrast, EME were considerably lower under favorable spring climatic conditions in the Mediterranean garden. This result, when evaluated with a previous Belgium study performed in a rather mild climate Previous glasshouse experiments suggest that experimental conditions influence EME expression, e.g. competition and light S. inaequidens, climate may represent a particularly predictable parameter compared to known dispersal distances field seed mass variation). Monty and Mahy Parental effects are unlikely to improve offspring fitness unless the environment is predictable relative to the maternal environment S. inaequidens, one of the most rapid plant invaders in Europe, relies on concomitant and not mutually exclusive mechanisms for successful invasion of distinct climates, even at a regional scale. Consistent with recent advances in invasive plant evolution research local adaptation vs. phenotypic plasticity” question, frequently addressed in the literature on invasive plant trait variation in the invaded range The results of this study indicate that As a result, if understanding the mechanisms responsible for phenotypic variation in relation to an environmental factor is the goal, not only is a reciprocal experiment preferred to single gardens, but sowing is also preferable to transplantation. In addition, garden experiments coupled with genetic analyses provide a more robust analysis. This represents a research opportunity for ecologists to understand how invasive plant populations face gradual environmental changes during colonization.Table S1Senecio inaequidens populations sampled in each climatic zones, along with the number of plants sampled for the AFLP analysis (n), the genetic variation (H), and the rarity index (DW).Geographic characteristics of (DOC)Click here for additional data file.Table S2Monthly climatic data during the experiment (March–December 2008) in both experimental gardens, based on climatic stations near the experimental sites.(DOC)Click here for additional data file.


Biological Conservation, Biotropica, Conservation Biology) and found limited application of high-resolution imagery in research, with only 26.8% of land cover studies analyzing satellite imagery, and of these studies only 10.4% used imagery ≤5 m resolution. Our results suggest that high-resolution imagery is warranted yet under-utilized in conservation research, but is needed to adequately monitor and evaluate forest loss and conversion, and to delineate potentially important stepping-stone fragments that may serve as corridors in a human-modified landscape. Greater access to low-cost, multiband, high-resolution satellite imagery would therefore greatly facilitate conservation management and decision-making.Technological advances and increasing availability of high-resolution satellite imagery offer the potential for more accurate land cover classifications and pattern analyses, which could greatly improve the detection and quantification of land cover change for conservation. Such remotely-sensed products, however, are often expensive and difficult to acquire, which prohibits or reduces their use. We tested whether imagery of high spatial resolution (≤5 m) differs from lower-resolution imagery (≥30 m) in performance and extent of use for conservation applications. To assess performance, we classified land cover in a heterogeneous region of Interior Atlantic Forest in Paraguay, which has undergone recent and dramatic human-induced habitat loss and fragmentation. We used 4 m multispectral IKONOS and 30 m multispectral Landsat imagery and determined the extent to which resolution influenced the delineation of land cover classes and patch-level metrics. Higher-resolution imagery more accurately delineated cover classes, identified smaller patches, retained patch shape, and detected narrower, linear patches. To assess extent of use, we surveyed three conservation journals ( Since the 1972 launch of the Earth Resources Technology Satellite (renamed Landsat 1), remotely sensed imagery has been increasingly used to monitor Earth’s ecosystems Satellite imagery employed in conservation research ranges from 1000 m to <1 m in resolution Previous comparisons of land cover classifications based on imagery of varying spatial resolutions  have revealed mixed results, with one type of satellite imagery failing to consistently perform best across different studies and systems We assert that conservation research would benefit from a better understanding of satellite imagery performance. The ability to detect forest disturbance and degradation (e.g. as a result of selective logging) with satellite imagery varies greatly The Atlantic Forest of South America is one of the major global “hotspots” for biodiversity due to its large number of endemic species and its dramatic decrease in size due to human-induced habitat loss and fragmentation We obtained cloud-free, geometrically corrected, and orthorectified IKONOS  and Landsat  imagery for the study region. Both imagery types spanned a 43,200 ha area that contained forest patches within the reserve, as well as the surrounding heterogeneous matrix. We selected Landsat imagery because it is readily and freely available, and frequently used for land cover classification k) for the overall classification and for each of the six land cover classes We collected 250 ground-truth points in the study area in June 2010 to serve as training samples for the classification; these points were taken throughout the private reserve. Using ERDAS Imagine 10, we drew polygons around each point and assigned the polygon to a land cover class. Training samples (number of locations and number of pixels) were distributed evenly across classes, with the exception of water because this class was limited in its spatial distribution across the study area (<1% of study area). We used the same 250 locations for both IKONOS and Landsat classifications. We used transformed divergence We converted raster data to vector data in ArcGIS 9.3 to calculate patch-level metrics. Continuous pixels were combined into a single vector patch, allowing for the quick delineation of number of patches and their sizes. We converted raster data to vector data instead of using object-based landscape analysis A grid consisting of 36 rows and 12 columns (432 1-km×1-km grid cells) was overlaid on both IKONOS- and Landsat-derived classifications . For eacWe calculated patch-level fragmentation metrics  using Patch Analyst 5.0 Linear habitat features, such as riparian corridors, have been found to be important conduits for species movement, thus, facilitating landscape-level connectivity Biological Conservation, Biotropica, and Conservation Biology) to assess the extent to which satellite imagery is currently utilized in conservation research. We reviewed all articles published in these three journals for the past two completed calendar years (January 2011 to December 2012), examining the methods to determine if satellite imagery was used in analyses. We did not include book reviews, editorials, or letters commenting on previous articles, and we limited our analysis to studies examining terrestrial (non-marine) patterns. For those studies utilizing remotely sensed data, we documented the satellite type , the spatial resolution of the imagery, the type of analysis , and the scale of analysis .We conducted a literature review of three top-tier conservation journals (k = 0.89) and 87.9% for Landsat (k = 0.86). For both IKONOS and Landsat, kappa statistics were lowest for the wetland class . The sample within a section of Reserva Morombi and its environs were dominated by forest, followed by agriculture, based on both IKONOS and Landsat classifications , with significant differences in cleared , water , and wetland  classes. Land cover based on IKONOS resulted in a greater number of patches overall for all six cover types  relative to Landsat.Overall classification accuracy was 90.3% for IKONOS , patch edge , shape index , perimeter-area ratio , mean distance from patch centroid to all other patch centroids , and distance from patch centroid to closest patch’s centroid . When we included only forest patches ≥0.5 ha, significant differences existed between IKONOS and Landsat in three of the six patch-level metrics: shape index , perimeter-area index , and mean distance from patch centroid to all other patch centroids .In total area, forest comprised 43.2% of the IKONOS image and 39.8% of the Landsat image, with a difference of 1473.6 ha. Although total forest area did not differ greatly between the two classifications, there were significant differences between IKONOS and Landsat in all six of the patch-level metrics we tested when analysis included all forest patches : patch at = 6.25, df = 39, p<0.001), and was correlated with the width of the forest fragment for both IKONOS  and Landsat . For example, unlike in the IKONOS classification  and delineated 55 times more forest patches than did Landsat, most of which were <0.5 ha. IKONOS correctly detected 95% of linear forest fragments 3.5 m –100 m in width, while Landsat detected only 45% of these same patches . IKONOS fication , long, lfication . Only ∼3Biological Conservation, Biotropica, Conservation Biology), of which 14.8% used land cover data in analyses. Of these 157 articles using land cover data, 26.8% used primary satellite imagery, while 73.2% used paper maps, aerial photos, Google Earth, or land cover data previously published by other authors. Of the 42 studies that analyzed satellite imagery, mean resolution was 84.4 m, with 66.7% and 10.4% using imagery ≥30 m and ≤5 m, respectively  and 10% used SPOT (launched in 1986) . Of 33 sDifferences in satellite imagery resolution are not trivial, and can manifest into stark differences in land cover classifications and resulting patch-level metrics . Ultimately these discrepancies are likely to influence interpretations of fragmentation patterns of a landscape, which can directly impact species and ecosystem modeling and conservation management plans. Many types of satellite imagery are available to conservation practitioners, but based on our literature review, most current conservation research does not take full advantage of either high-resolution or low-resolution imagery. Although high-resolution imagery can be difficult to obtain, primarily due to cost Biological Conservation, Biotropica, and Conservation Biology revealed that more than 70% of studies quantifying land cover used previously published material, aerial photos, paper maps, or Google Earth as their main resources, instead of satellite imagery (of any resolution). When satellite imagery was used, Landsat (30 m resolution) was most common  may not substantially increase, higher-resolution imagery is more likely to discern small and linear habitat features, which may be vital to landscape connectivity. Given that human activities have caused the conversion of more than 50% of the world’s terrestrial surface Additionally, high-resolution imagery will be valuable for more precise evaluation of habitat area and edge in landscapes. While species-area relationships have been widely used for conservation Classification using IKONOS imagery improved the delineation of forest fragments, with smaller mean size than fragments delineated using Landsat imagery. These differences were not surprising, however, given the smaller pixel size of IKONOS. Unexpectedly, however, the IKONOS-based classification led to a delineation of fewer forest patches (229) than did Landsat (237 patches) for patches ≥0.5 ha. While a difference of 8 patches may seem trivial, a classification using higher resolution imagery is expected to result in a greater number of patches (not less), due to a greater ability to distinguish smaller features. In contrast, we found that Landsat missed detecting several small forest fragments and narrow riparian corridors that connected other habitat patches, thus resulting in a classification of more disjunct, parcellated patches instead of one larger, interconnected patch. These findings have important consequences when evaluating fragmentation effects, especially in the case of heterogeneous and complex landscapes where habitat patches may be irregular and/or linear in shape, and where corridors may be critical for the movement of individuals and populations of a variety of species.Although our findings are site-specific to eastern Paraguay, like Masuoka et al. Although free data sets and free satellite imagery are available online High-resolution imagery, as well as long-term, time-series data (e.g. Landsat), are essential for conservation, economic, and social research and applications


Solanum, section Petota) are adapted to multiple habitats along the Andes, and natural hybridizations have proven to be a common feature among species of this group. Solanum× rechei, a recently formed hybrid that grows sympatrically with the parental species S. kurtzianum and S. microdontum, represents an ideal model for studying the ecologically and evolutionary importance of hybridization in generating of epigenetic variability. Genetic and epigenetic variability and their correlation with morphological variation were investigated in wild and ex situ conserved populations of these three wild potato species using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. We observed that novel methylation patterns doubled the number of novel genetic patterns in the hybrid and that the morphological variability measured on 30 characters had a higher correlation with the epigenetic than with the genetic variability. Statistical comparison of methylation levels suggested that the interspecific hybridization induces genome demethylation in the hybrids. A Bayesian analysis of the genetic data reveled the hybrid nature of S. × rechei, with genotypes displaying high levels of admixture with the parental species, while the epigenetic information assigned S. × rechei to its own cluster with low admixture. These findings suggested that after the hybridization event, a novel epigenetic pattern was rapidly established, which might influence the phenotypic plasticity and adaptation of the hybrid to new environments.Interspecific hybridization is known for triggering genetic and epigenetic changes, such as modifications on DNA methylation patterns and impact on phenotypic plasticity and ecological adaptation. Wild potatoes ( Spartina  constitute an ideal model to analyze the evolutionary consequences of hybridization in the generation of epigenetic variation and its significance in the phenotypic plasticity and ecological adaptation: (i) Natural hybridization and hybrid speciation have been proposed to be prevalent in this group of plants  wis Hawkes ; and  is one of the most extensively documented  and Solanum microdontum (Bitter) also grow sympatrically (Solanum kurtzianum is a diploid species (2n = 2x = 24) that grows on four Argentinean provinces, along a transect approximately 900 km long  populations have been recorded  accessions of these species provided by the Potato and Forages Germplasm Bank Instituto Nacional de Tecnología Agropecuaria, Balcarce, Argentina  Plants from wild populations of Thirty qualitative and quantitative characters were measured in three plants per accession once flowering had started . Morphol−1.DNA was extracted from fresh leaves following Dellaporta et al.  with an EcoRI and MseI were used as the rare and frequent cutter, respectively. The restriction reaction contained 2 units of EcoRI, 2 units of MseI, 1.25 μL of buffer 2 (NEB), 100 ng·μL−1 BSA, and 250 ng of DNA in a final volume of 12.5 μL and was incubated for 3 h at 37°C. The ligation reaction contained 5 pmol of EcoRI adaptors, 50 pmol of MseI adaptors, 1.25 μL T4 DNA ligase buffer (Promega), 0.75 units of T4 DNA ligase , and 6.25 μL of the digested products in a final volume of 12.5 μL. Adaptors were ligated for 3 h at 20°C. Preselective amplification was accomplished using [EcoRI + A] and [MseI + C] primers. Each PCR contained 40 ng of each primer, 400 μmol of dNTPs, 1× Taq DNA polymerase buffer, 1 unit of Taq DNA polymerase (Invitrogen), and 1 μL of the ligation products in a final volume of 25 μL. The amplification profile was as follows: 20 cycles at 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min. The pre-selective PCR products were diluted 1:3 with sterile ultra-pure H2O. Selective amplification was performed with fluorescence-labeled EcoRI-selective primers and unlabeled MseI selective primers , 0.5 units of Taq DNA polymerase (Invitrogen), and 5 μL of the diluted pre-selective product in a final volume of 20 μL. The PCR protocol was as follows: 14 cycles at 94°C for 30 s, 65°C for 30 s (decreasing 0.7°C per cycle), and 72°C for 1 min, followed by 23 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min.To assess genetic variability in the evaluated material, an AFLP analysis was performed as described by Vos et al. . EcoRI a primers . The reaAfter selective amplification, 1 μL of each sample was mixed with 8.5 μL Hi-Di formamide and 0.5 μL of the size standard Genescan 500HD ROX . Samples were then denatured at 90°C for 3 min and were run on an Applied Biosystems Genetic Analyzer 3130. Semi-automated scoring was performed on the resulting electronic profiles using GeneMapper version 3.7 (Applied Biosystems).MseI by HpaII and MspI, while the rare cutter remains unchanged. HpaII and MspI are isoschizomers which recognize the same tetranucleotide restriction site (5′-CCGG), but have different sensitivities to methylation at cytosines. HpaII is sensitive to full methylation (both strands methylated) of either cytosine but cleaves the hemimethylated external cytosine, whereas MspI is sensitive only to methylation of the external cytosine. In this technique, the presence of the fragments in both profiles indicates a non-methylated site, while the absence of these fragments in both amplifications is ambiguous. It could be due to complete site methylation or to variation in the nucleotide sequence  with 100 ng·μL−1 BSA and 1× buffer H (Promega) in a final volume of 20 μL. To ensure complete digestion, incubation was performed at 37°C over night. The digest was split into two equal volumes and 10 U of HpaII (Promega) and 10 U of MspI (New England Biolabs) were added to each half, along with 100 ng·μL−1 BSA and the appropriate buffers in a final volume of 20 μL for the second digestion. Ten μL of the digested solution was ligated with 25 pmol of each EcoRI and HpaII/MspI adaptors, using 0.75 units of T4 DNA ligase (Promega) in a final volume of 20 μL. Adaptors were ligated for 3 h at 15°C. Pre-amplification was performed using 1 μL of the ligation products and 4 pmol of the [EcoRI + 0] and [HpaII/MspI + A] primers in a final volume of 20 μL containing 1× PCR buffer, 0.1 mmol·L−1 dNTPs, and 1 U Taq polymerase (Invitrogen). The pre-amplification products were diluted 1:3, and 1 μL was used in the selective amplification reaction with the EcoRI- and HpaII/MspI-selective primers  a methylation-sensitive polymorphisms matrix, with fragments that differed in the presence/absence patterns between the EcoRI/HpaII and the EcoRI/MspI digests in at least one genotype. In this case, patterns were codified from 0 to 3  or absent (0). For the MSAP analysis, two different matrices were built: (i) a binary matrix with methylation-insensitive polymorphisms, loci where the fragments were either present or absent in both m 0 to 3 , and theFor both AFLP and MSAP procedures, a subset of 23 samples were replicated starting from the same DNA extractions, to calculate the error rate of the techniques. Loci with different patterns on replicates were excluded from analysis as possible methodological artifacts.S. microdontum and S. kurtzianum . ANOVAs were performed to compare fragments per individual and methylation percentages between species using STATGRAPHICS Centurion XVI software.The total number of fragments (loci) for AFLP and MSAP was computed for each of the three species. Common and species-specific fragments were identified, and the mean number of fragments per individual was also calculated. Based on MSAP data, percentages of methylated, hemimethylated, and unmethylated loci were determined for each species and for a combination of d = 1–s), and a mean distance was calculated for each species. ANOVAs were carried out to compare genetic and epigenetic variability among species using STATGRAPHICS Centurion XVI software.Multivariate analyses were carried out separately for genetic and epigenetic data sets. PCoA and cluster analyses were performed to determine relationships among individuals based on genetic and epigenetic polymorphism. Similarity matrices were generated based on the Dice coefficient, and the UPGMA linkage method was used for dendrogram construction. Also, the Mantel test was carried out to contrast genetic, epigenetic, and morphological matrices. For this, matrices containing shared individuals were constructed and similarity matrices, and tree topologies were compared using 1000 permutations. PCoA, cluster analysis, and the Mantel test were performed with NTSYS-pc 2.11, and for bootstrapping analysis, the WinBoot program was used . Assuming that allele frequencies within populations follow a Dirichlet distribution, BayeScan calculates locus-population FST coefficients. Selection is introduced by decomposing locus population FST coefficients into a population-specific component β (beta), shared by all loci, and a locus-specific component α , shared by all the populations using a logistic regression. Departure from neutrality at a given locus is assumed when the locus-specific component is necessary to explain the observed pattern of diversity (α significantly different from 0). A positive value of alpha suggests diversifying selection, whereas negative values suggest balancing or purifying selection. This leads to two alternative models for each locus, including or not the alpha component to model selection. BayeScan implements a reversible-jump MCMC algorithm to estimate the posterior probability of each one of these models. Posterior odds (PO) are simply the ratio of posterior probabilities and indicate how more likely the model with selection is compared with the neutral model  >1, which provides decisive support for acceptation of the model according to Jeffreys' scale of evidence  following the manufacturer's protocol. Direct sequencing of the fragments was performed by Sanger technology in an Applied Biosystems Genetic Analyzer 3130XL. Sequences were edited and analyzed with BioEdit 7.0.9 Hall .http://www.jcvi.org/; http://blast.jcvi.org/euk-blast/plantta_blast.cgi).Analysis of DNA similarity was carried out using the BLASTn packages implemented at The Institute for Genomic Research  and horticultural orchards. The parental species, S. kurtzianum and S. microdontum, were not present in these disturbed areas. We also explored natural environments for the presence of the three species. In the gorge of the Pismanta River, along a transect approximately 1 km long, we sampled six populations of S. × rechei . Then, we explored several gorges in the road to Mina de Oro, and only one population of S. × rechei was sampled (GV12). We collected four populations of S. kurtzianum separated from the area of hybridization by a distance of about of 5 km (CG) and 50 km . In 2010, we expanded the collection area along the gorge of the Pismanta River in a transect of about two kilometers long. Again, we found that the species with greater distribution was the hybrid S. × rechei and sampled one more population (CMR). In addition, we found and sampled one additional population of S. microdontum (CMM) and one population of S. kurtzianum (CMK) within the gorge of Pismanta River.We collected plant material from the hybridization area, in the locality of Guanchín (La Rioja), in two opportunities. In the 2008 expedition, we found that the most common species was the hybrid × rechei . No popuS. kurtzianum and S. microdontum, showed no significant differences in four  additional characters.We measured 30 qualitative and quantitative characters in three plants per accession once flowering had started  to test S. kurtzianum and S. microdontum were analyzed (22 of 30). Seven characters resulted intermediate and 15 resulted non-intermediate (P > 0.05), and the hypothesis of divergence could not be rejected. Six of the non-intermediate characters were transgressive in S. × rechei beyond the parental species.For the character count procedure, only those that significantly differed between rmediate ; thus, tS. microdontum and S. kurtzianum by their morphotype were all gathered into two well-defined groups. On the other hand, individuals classified as S. × rechei formed three different groups: two located close to each other and between S. kurtzianum and S. microdontum, and a third group overlapped with S. kurtzianum group. In the cluster analysis, S. × rechei individuals were distributed in a main cluster and in a second cluster mixed with S. kurtzianum individuals, while S. microdontum formed a separated group  was significantly (P < 0.01) higher than that for the mid-parental value (Mean = 44.7%). The percentage of methylated loci for S. × rechei (Mean = 40.5%) was significantly (P < 0.05) lower than that for the mid-parental value (Mean = 43.9%). The difference in the hemi-methylation level was not statistically significant  are unequivocally methylation changes; as for the same loci, the parental species showed the presence of the fragments, but with a different pattern respect to the hybrid. For the other 14 loci (4%), the parental species showed no fragments, which could indicate both a change in the methylation status in the hybrid (if the sites where hypermethylated in the parents) or a genetic variation.The Venn diagram in P > 0.05), although the comparison of genetic against epigenetic distances within species was statistically significant for the three species  were found between species  than with AFLP . MSAP matrix holds a greater correlation with AFLP (r = 0.78) given that both are methodologically related .The Mantel tests  showed aK = 2 and 3 presented similarity coefficients close to 1, which means that individuals were repeatedly assigned to the same groups in all runs. This behavior was also observed in the bar plots. With MSAP data, the similarity coefficient was 1 only for K = 3. Furthermore, the ΔK values suggested K = 2 as the optimal for AFLP and K = 3 for MSAP  > 1) on the Jeffreys' scale. Fifteen of these markers were pointed out “very strongly” to be under selection (log10(PO) > 1.5), and for seven of them, evidence was considered “decisive” (log10(PO) > 2; ST coefficients and positive α values and are therefore interpreted as having been affected by divergent selection.BayeScan analysis identified 24 MSAP epiloci under selection (or linked to selected loci), with “strong evidence”  to an EST (Expressed Sequence Tag) from Solanum chacoense (GeneBank Accession DN978520). The other fragment of 125 bp was similar  to a sequence of Solanum lycopersicum (GeneBank Accession TA55928_4081) annotated as a pararetrovirus-like sequence reported in tobacco.Four MSAP fragments with the highest FS. × rechei based on a genome-wide screening method. The hybrid nature of S. × rechei had been analyzed using morphological and molecular (RFLPs) approaches by Okada and Hawkes  used by Clausen and Spooner (S. microdontum from Guanchín (La Rioja province) growing sympatrically with S. × rechei. In S. microdontum, PcoA analyses of genetic data showed intraspecific discontinuities, separating populations from La Rioja province in one group and the populations from Catamarca and Salta provinces in another group. However, these differences were not supported in the Bayesian STRUCTURE analysis. This result confirms that S. microdontum material used by Clausen and Spooner , S. × rechei presented genotypes with different floral abnormalities, suggesting that recently formed hybrids may suffer genetic and epigenetic instability as a result of the interspecific hybridization event.As described by Marfil et al.  for a diSpartina spp.  and loss (4%) of AFLP fragments were observed in the hybrid, possibly as a result of genome rearrangements induced by the interspecific hybridization. Similar results were described in interspecific hybrids of wheat  with the epigenetic than the genetic information  with individuals displaying admixture between S. kurtzianum and S. microdontum. Instead, the epigenetic information clearly assigned S. × rechei individuals to a third cluster (K = 3) with high probability (Dactylorhiza, Paun et al. (S. × rechei the hybridization event rapidly established a novel epigenetic pattern, different from the parental species, which could modulate the regulatory networks and persist stably over many generations possibly through clonal propagation by tubers. According to our results, the adaptation of S. × rechei to grow as a weed in disturbed environments, such as along roadsides or under trees in orchards (Okada and Hawkes ST values and therefore interpreted as having been affected probably by divergent selection. Nevertheless, it is possible that these epialleles may have arisen as a consequence of the hybridization event and have been maintained by clonal propagation. One of the sequenced epialleles was related to an endogenous pararetrovirus sequence reported in tobacco, possibly conferring heritable virus resistance (Mette et al. Nicotiana tabacum and the diploid N. sylvestris. These results suggest that the epiallele was selected in both species and conferred advantageous traits to tobacco and N. sylvestris. It is likely that the same epiallele was under selection in the potato species studied.Our study showed a high level of genetic and epigenetic polymorphism for the three taxa analyzed, where 98% and 99% of the AFLP and MSAP loci were polymorphic, respectively. For each of the studied taxa, the epigenetic variability was significantly higher than the genetic variability, based on Dice coefficient . PcoA anthan 0.5 . These rbability , with sen et al.  showed tS. kurtzianum and triploid cytotypes of S. microdontum, some hybrid genotypes are likely to be aneuploids. However, Okada and Hawkes (Changes in chromosome number could play a role in the origin of the variability naturally found in the hybrid populations. As the hybrids were probably originated by the hybridization of the diploid d Hawkes  found onSolanum species from section Petota are the result of hybridization. There is also an important amount of phenotypic plasticity in this section (Correll Solanum species section Petota and that the analysis of the epigenetic variation may shed some light on the intricate taxonomy and systematic relationships among species of this section.Hybridization is an important event in the evolution of the potato species, and several authors suggested that more than 10% of the  Correll . Our res Correll , given tLocation, AFLP, and MSAP data: Dryad entry doi:10.5061/dryad.q6s27.


FST, routinely underestimated genetic differentiation by a factor three to four compared to the equivalent statistic Jost's Dest (FST statistics, we argue that these could be useful to identify and quantify qualitative differences between populations, which are important from management and conservation perspectives as an indicator of existence of biologically significant variation among tributary populations or a warning of critical environmental damage.Little is known about the microevolutionary processes shaping within river population genetic structure of aquatic organisms characterized by high levels of homing and spawning site fidelity. Using a microsatellite panel, we observed complex and highly significant levels of intrariver population genetic substructure and Isolation-by-Distance, in the Atlantic salmon stock of a large river system. Two evolutionary models have been considered explaining mechanisms promoting genetic substructuring in Atlantic salmon, the member-vagrant and metapopulation models. We show that both models can be simultaneously used to explain patterns and levels of population structuring within the Foyle system. We show that anthropogenic factors have had a large influence on contemporary population structure observed. In an analytical development, we found that the frequently used estimator of genetic differentiation, t's Dest . These s Salmo salar). The spatial scales of these studies have varied from intercontinental . Given the lack of consensus as to which evolutionary model best explains patterns of genetic differentiation in wild Atlantic salmon populations, and that studies focusing on within-river genetic structure in this species remain relatively scarce, more studies are warranted.The River Foyle system, in the northwest of Ireland, was, until recently, one of the most productive wild Atlantic salmon fisheries in Western Europe. Annual commercial catches of salmon in the estuary and the adjacent sea area have ranged between 12,176 and 44,000 salmon between 1997 and 2006 , while aThe Foyle system, in contrast to the Moy, Varzuga, Teno, and Sainte-Marguerite , is a teThe specific aims of this study are to: (1) determine spatial genetic structure in the Foyle system, (2) asses which evolutionary model (member-vagrant or metapopulation) best fits the observed results, and (3) compare and discuss results with other such studies from the recent literature.2. The main tributaries of the Foyle are the rivers Finn and Derg to the west, and the Owenkillew River in the eastern part of the catchment. The Foyle main stem is known under different names throughout its course. Downstream of the confluence of the Drumragh River and the Camowen River, the main stem is called River Strule until it meets the River Derg downstream of which the main stem is known as River Mourne. After the River Finn joins the River Mourne, the main stem is called River Foyle until it drains into Lough Foyle. The Faughan River does not join the Foyle main stem, but directly enters Lough Foyle to the east of the main river.The Foyle River system  is located in the northwestern corner of the island of Ireland, forming part of the land border between Northern Ireland (United Kingdom) and the Republic of Ireland . The Foy+ (fry) and 1+ (parr) fish. A total of 13 sampling sites were visited. Among these, two temporal replicate samples originally taken at two sampling sites (Deele and Faughan Rivers) in 1999 were included to test for temporal variation. In total, 1086 individual tissue samples were collected for this study . The aim was to collect 96 samples per sampling location. At some of the  tributaries, this number was not reached due to smaller numbers of juvenile salmon present. In these cases, the number of samples taken was reduced to 48.Sampling was conducted throughout the Foyle system, including the adjacent River Faughan, with an average waterway distance between the sampling locations of 72 ± 28 (SD) km and a range of 17.8–132.4 km. Following consultation with local fishery managers, sample coverage included most known spawning and nursery areas in all major tributaries. Sampling by electrofishing conducted during 2006 and 2007 captured juvenile Atlantic salmon from multiple year classes; 48 or 96 fish per sampling location with ideally a 50/50 ratio of 0is study ; Table 2The sampling of multiple year-classes combined with taking the samples from river stretches of several hundred meters minimized the likelihood for the “Allendorf-Phelps effect” , wherebyTissue samples from the juvenile Atlantic salmon were taken by removing the adipose fin before releasing the fish in the case of the parr samples, or by sacrificing the whole fish for the fry samples. In both cases, the tissue samples were preserved in molecular grade 99% ethanol until analysis.2) of fin tissue. Extracted DNA was kept frozen at –20°C until amplification using a polymerase chain reaction (PCR). Samples were screened for a suite of seven highly polymorphic microsatellite loci; Sssp1605, Sssp2201, Sssp2210, Sssp2216, SsspG7  protocol  on small, SsspG7 , Ssa197,, SsspG7 . The for2, 0.3 µM forward and reverse primer (0.15 µM in the case of Ssa197), 0.13 U Taq DNA polymerase (Invitrogen), and ultraPURE H2O  to a total reaction volume of 15 µl. For the remaining four loci, the PCR reaction consisted of 1 µl of extracted template DNA, 0.2 mM dNTP's (Invitrogen), 1× reaction buffer , 5 mM MgCl2, 0.38 µM of the forward and reverse primer for Sssp1605, 0.95 µM for Sssp2201, 0.17 µM for Sssp2210, 0.88 µM for Sssp2216, 0.1 U of Taq DNA polymerase, and ultraPURE H2O to a total reaction volume of 10 µl. The PCR, in which Sssp2210/Sssp2201 and SsspG7/Ssa197 were multiplexed, was performed using the following amplification cycles: denaturing at 95°C/2 min followed by 33 cycles of 95°C/45 sec, 58°C/45 sec, 72°C/45 sec, which was followed by a final cycle of 95°C/45 sec, 58°C/45 sec, 72°C/5 min on a Thermo Hybaid PCR Express thermocycler .The individual PCR amplification reactions for loci SsspG7, Ssa202, and Ssa197 consisted of 1 µl of extracted template DNA, 0.2 mM dNTP's , 0.7× reaction buffer , 1.5 mM MgClThe PCR products were prepared for electrophoresis on an ABI 3130 Automatic DNA Analyzer  according to the manufacturer's instructions using ABI GS-500 LIZ internal size standard. Alleles were scored and sized using the automated bin function of the ABI GeneMapper v4.0 software, but were manually checked and verified.Microchecker v2.2.3  was usedNA), allelic frequencies, observed (HO), and expected (HE) heterozygosity, using the program GENEPOP v3.4  and between samples Deele and Faughan and their 1999 equivalents, was assessed by testing for pairwise heterogeneity in allelic frequency distributions between temporal samples with GENEPOP v3.4. The same program was also used to test for genotypic linkage disequilibrium across putative populations, to confirm independent allelic segregation among different marker loci.FST values, or variance in allele frequencies (AR), which is the allele number per population corrected for differences in sample size. Corrections to account for multiple tests, sequential Bonferroni correction,  as implemented in the programme POPULATIONS v1.2.30beta  Chord distance measure , we followed K) based on the rate of change in the log probability between consecutive K values, which ranged from K = 1 to K = 20 with a burn-in and Markov chain Monte Carlo length of 10,000 each  for 20 runs per K-value. The modal value of the ΔK distribution is assumed to represent the true number of clusters or populations in the data. The reliability of the replicate analysis mentioned above was enhanced by use of the CLUMPP 1.1.2 (K)] of the data as the most likely value of K  implemented with the software Arlequin 3.1.1 , only five genotyping errors were found; a genotyping error rate of 0.3%. Three of these errors were allelic dropouts, the other two either false alleles or contaminations. The error rate in this study was low see  and therNo statistically significant differences in genetic composition between the two cohorts sampled in 2006 and 2007 were observed in any of the sampling locations or between the temporally spaced Faughan and Deele samples. Thus, all temporal samples representing all 13 sampling locations were pooled prior to subsequent analyses.The mean number of alleles per locus varied between 12.9 in sample Owenreagh East and 18.4 in sample Faughan. The average expected heterozygosity  was veryFST and Dest between sampling locations, ranged from 0.001 to 0.06 for FST and from 0.003 to 0.3 for Dest  + var(D)(1/A)3] where A is the arithmetic mean and var(D) is the variance of Dest values over loci) was 0.12 . Despite ongoing debate over the application Jost's D, and the merits of using FST for microsatellite data, we found a near-perfect correlation between both estimates  in the present dataset. Interesting, while we also found a strong positive correlation between DC and Dest , this was not as strong as between FST and Dest.Genetic differentiation, estimated as pairwise for Dest . In geneDC) genetic distance measure . This observed overall positive IBD pattern, however, was strongly influenced by the major regions . Within each individual region, there was no evidence of IBD, with the exception of the western regional group. In the latter case, however, given small number of samples within the group, results should be interpreted with caution. To test the independent effects of both geographical distances and the major geographical regions, as identified by the previous analyses , in the patterns of population differentiation observed, partial Mantel tests were carried out (FST and Dest) were found to be significantly correlated to both geographical distances and regions, with the latter variable having a more significant effect. Together, these analyses provide unambiguous evidence for IBD in the River Foyle among regions, but not within all regions per se.There was significant overall positive association between geographical distance and ried out . GeneticK was found to be bimodal at K = 3 and K = 4. Close examination of the graphical output of the STRUCTURE analyses .The results of the STRUCTURE analysis are in agreement with the Chord genetic distance clustering in supporting three major genetic groups within the Foyle catchment, which are generally defined by geography . Interestingly, at the most basic level of structure, Δanalyses  suggestsanalyses . The resP < 0.001) percentage of the observed genetic variance is explained by differences among samples within (1.35%), and among regions (0.98%), thus confirming previous analyses.The results of the AMOVA analysis, carried taking in consideration (1) the main regions within the Foyle System ; (2) samples within regions; and (3) samples within the Foyle System, are summarized in The overall levels of genetic variability in salmon of the Foyle River system  reported in this study are at the high end of the range reported for similar population genetic studies using microsatellite markers on Atlantic salmon e.g., . This isSimilar to previous studies examining within river system population structuring e.g., , substanDest relative to FST are in line with FST (GST) tends to underestimate true levels of population differentiation compared to Jost's D and related statistics . Thus, there is an inherent methodological penalty associated with the use of this class of markers with this statistic. Many authors, however, still fail to acknowledge or recognize this important issue, with the unfortunate consequence that significant genetic differences are often dismissed as being inconsequential on the basis of “small”FST estimates. Both FST estimates in relation to the evolutionary factors they are measuring, for example, gene flow, mutation, random genetic drift, and selection . We argue, however, that levels of microsatellite polymorphisms observed in the Steller's sea lion  are comparatively low in relation to what is observed in other species . Thus, the bias associated with high levels of genetic diversity as discussed by In instances where these “adjusted” measures of genetic differentiation have been implemented, results have been inconclusive. For instance, FST, as recommended by Jost's (2008)Dest should provide a more appropriate measurement of population differentiation. Indeed, we suggest that reviews should be carried out to reassess the levels and patterns of population divergence from studies solely relying on uncorrected FST. This should be a priority for valuable Atlantic salmon populations where the ability to accurately identify individual population units is critical. Furthermore, from a conservation perspective, it would be relevant to inform managers about the caveats of the interpretation of standard FST previously estimated and presented insofar as microsatellite data are concerned.As conservation and management strategies of many salmonid fish species have been predominantly based on population differentiation estimates of FST values (FST = 0.02) did not differ greatly. For instance, FST values of 0.024 for the Moy River in the west of Ireland. Dest estimates, in the Foyle, both over all loci and population samples were on average three to four times higher compared to global FST values. Again, using the argument above, if this study had been limited to only using FST as a measure of genetic differentiation, the vast majority of differentiation between samples could have been perceived as very small. Given the argument above, the important point is whether the statistic is significant or not, while the value itself is largely irrelevant.Notwithstanding the issue discussed above, for a comparative perspective between this and other genetic studies on Irish Atlantic salmon populations using similar microsatellite markers, it is worth noting that the global uncorrected Given observed differences in allelic frequency distribution (exact tests), the majority of the samples examined from the Foyle represent more or less independent temporally stable population units . The only exception  involved the geographically close sampling locations Finn and Reelan  in the Foyle catchment, the overall pattern of which generally accords with the geographical and hydrological organization of the catchment, and which is consistent with a member-vagrant evolutionary model. However, while the membership of samples belonging to the West group is geographically well defined, some inconsistencies were observed in the membership of the Northeastern and Southeastern groups. We argue below that these discrepancies can be explained by anthropogenically mediated disturbances.The western tributaries comprise the largest rivers in the Foyle catchment with correspondingly large population sizes. In comparison to the other areas of the catchment, the western rivers are considered to be reasonably pristine from an environmental perspective. This area of the catchment is also well known for the presence of multi-sea winter fish, a life-history trait that is uncommon in the rest of the catchment. Thus, the genetic uniqueness of samples from this area is likely to reflect an independent evolutionary history. Overall, there was no strong evidence for IBD within groups, which suggests high levels of conductivity and therefore is in agreement with the concept of a metapopulation evolutionary model.Within the Northeast group, the most northern tributaries , similar to those in the west of the catchment, are also large, with good salmon habitat and supporting large populations. Indeed, available demographic data for the Foyle system (authors’ unpublished data) suggest that the tributaries from the north and from the west of the catchment are responsible for the most of the current salmon productivity in the Foyle . In contrast, however, the population sizes of the eastern  and southeastern (Owenreagh South and Drumragh) tributaries, despite their large habitat area (between 25% and 35% of the catchment) are relatively narrow and small (authors’ unpublished data). This part of the Foyle catchment is characterized by intensive farming and it is heavily impacted by arterial drainage schemes resulting in the disturbance of natural habitats, which severely limit productivity. Therefore, the genetic pattern characteristic of samples belonging to this region reflects random stochastic processes , associated with anthropogenetic impacts, such a loss of spawning habitats or poor water quality, as the main factors shaping contemporary population genetic structure in these regional groupings.This hypothesis is supported by the lack of evidence for substructuring as provided by both NJ and the STRUCTURE analyses. Alternatively, the observed similar genetic makeup of samples from both regions could reflect past historical gene flow prior to the development of the extensive arterial drainage scheme in the eastern and southern regions of the catchment. At the moment, the data are insufficient to disentangle both hypotheses.While we observed strong and statistically significant positive correlation between geographical distance and genetic distance (IBD) within the River Foyle Atlantic salmon stock, this trend was linked to the three indentified genetic regional groupings rather than individual samples. Interestingly, IBD within a single river system is relatively uncommon in Atlantic salmon. Evidence for IBD is more frequently reported from studies on Atlantic salmon population genetic structure at larger spatial scales often involving multiple river catchments e.g., .The Atlantic salmon stock of the River Foyle most likely exists as several distinct, long-term temporally stable, populations within well-defined geographical regions, in which migration occurs more frequently than between populations from different regions. It is clear from our findings that the genetic structure of the River Foyle Atlantic salmon stock is complex. Thus, it cannot be simply defined within a single evolutionary model. If considering the whole system, the population structure of salmon within the Foyle catchment is better explained under the member-vagrant evolutionary model as confirmed by the clear presence of IBD at this level. However, within each of the major genetic regions identified, the metapopulation model best explains the observed structuring, as there is no evidence for IBD at this level. We argue that these models are not mutually exclusive and can operate simultaneously on different geographical scales within a single river system. Thus, any attempts to identify a best model fit would be artificial and not reflective of the complex evolutionary history commonly associated with the species.Interestingly, From a conservation and management viewpoint, focus should be orientated toward identifying and preserving appropriate evolutionary entities. In the Foyle, using genetic evidence and an environment quality indicator (EQI), it is evident that conservation efforts should be targeted toward southern and eastern tributary catchments. This could be done by restoring the integrity of habitats prior to the development of intensive farming activities and associated drainage schemes. The data also suggest that the management of salmon in the Foyle should accommodate the three regional groupings identified in this study. Future studies should continue to explore the interplay between the evolutionary models determining genetic structure and to what extent the findings reported in here apply elsewhere.Genotypes and all other raw data used in this study are stored on an electronic database at AFBINI. At request available to others via the corresponding author.


Eurythenes gryllus is one of the most widespread amphipod species, occurring in every ocean with a depth range covering the bathyal, abyssal and hadal zones. Previous studies, however, indicated the existence of several genetically and morphologically divergent lineages, questioning the assumption of its cosmopolitan and eurybathic distribution. For the first time, its genetic diversity was explored at the global scale  by analyzing nuclear (28S rDNA) and mitochondrial  sequence data using various species delimitation methods in a phylogeographic context. Nine putative species-level clades were identified within E. gryllus. A clear distinction was observed between samples collected at bathyal versus abyssal depths, with a genetic break occurring around 3,000 m. Two bathyal and two abyssal lineages showed a widespread distribution, while five other abyssal lineages each seemed to be restricted to a single ocean basin. The observed higher diversity in the abyss compared to the bathyal zone stands in contrast to the depth-differentiation hypothesis. Our results indicate that, despite the more uniform environment of the abyss and its presumed lack of obvious isolating barriers, abyssal populations might be more likely to show population differentiation and undergo speciation events than previously assumed. Potential factors influencing species’ origins and distributions, such as hydrostatic pressure, are discussed. In addition, morphological findings coincided with the molecular clades. Of all specimens available for examination, those of the bipolar bathyal clade seemed the most similar to the ‘true’ E. gryllus. We present the first molecular evidence for a bipolar distribution in a macro-benthic deep-sea organism. The deep sea represents the largest ecosystem on our planet, yet our awareness of its fauna is remarkably recent, dating back to the Challenger expedition (1872–1876) e.g. . In the Many deep-sea species have geographic distributions encompassing one or more entire oceans as well as very wide bathymetric ranges e.g. . The assEurythenes gryllus , a giant bentho-pelagic deep-sea amphipod. Among all known lysianassoid species, E. gryllus has the widest geographical distribution. It occurs in every ocean and has a depth range from 550 to 7,800 m, covering the bathyal, abyssal and hadal zones An example of such presumably cosmopolitan species is E. gryllus was found to comprise several morphologically distinct and genetically divergent lineages, which seem to be vertically stratified However, E. gryllus at the global scale  using both nuclear and mitochondrial DNA sequence data. We tested for evidence of multiple species and we attempted to identify potential barriers in the deep sea that could isolate gene pools and allow populations to diverge into separate species. Finally, an analysis of morphological characters was performed to detect the possible existence of previously overlooked morphological differences within E. gryllus and to ascertain which of the genetic lineages, if any, is identical to the material described as E. gryllus.Aforementioned studies were generally carried out on a limited geographic scale and solely based on either morphology or genetic analyses using a single molecular marker. A study at a worldwide scale, based on a combined approach by both molecular and morphological tools has not yet been undertaken. Therefore, we explored genetic diversity in E. gryllus were collected during recent expeditions of RV Polarstern and RV Meteor. Traps were deployed at the following locations: the Arctic Ocean, Brazil and Argentine Basins and several locations in the Southern Ocean. Permission to collect amphipod samples from the prospected areas in the Southern Ocean was obtained from the Belgian authorities observing the guidelines of the Environment Protocol to the Antarctic Treaty (Permit nr. 01/10). None of the other sampling stations were within specially protected areas and no permits were required for work in these locations. Eurythenes gryllus is not an endangered or protected species. Sampling details are listed in et al.et al.E. gryllus.Specimens of et al.Genomic DNA was isolated from pereopod 6 using the NucleoSpin Tissue kit (Macherey-Nagel). PCR amplifications of the mitochondrial COI and 16S rRNA gene fragments were carried out using the universal primers LCO1490, HCO2198 Chromatograms were checked and sequence assembly was carried out with CodonCode Aligner v2.0.6 (Codon Code Corporation). COI sequences were aligned manually; 16S and 28S rDNA sequences with the MAFFT 6 web server (using the G-INS-i option) E. gryllus, using the combined dataset and 16S rDNA dataset. The combined dataset consisted of COI, 16S and 28S rDNA sequences of 47 specimens collected for this study. Identical sequences were collapsed into unique haplotypes, resulting in 45 concatenated sequences, which were used to perform parsimony and Bayesian analyses. Sequences of the lysianassoid Abyssorchomene spp., available from GenBank, were used to define the outgroup . The 16S rDNA dataset generated in this study (66 sequences) was complemented by sequences of E. gryllus available in GenBank: 96 sequences representing 12 different haplotypes from France and Kocher et al.Eurythenes sp. (U40449) were recognized as a distinct species by France and Kocher E. thurstoni in this study (according to Stoddart and Lowry Tree-construction methods were used to identify possible clades within E. gryllus from GenBank Relationships between haplotypes were investigated by generating haplotype networks with the complete 16S dataset (163 sequences) using TCS 1.21 sensu Hebert et al.Single gene datasets of the three genes were used for identifying species complexes from genetic divergence between specimens  was high: K2P distances ranged from 4.5 to 13.2% . FurtherUltrametric trees obtained for 16S sequences were subjected to the GMYC analysis. The number of ML clusters ranged from eighteen (single threshold method) to thirty-two (multiple threshold method) , which dEurythenes is known to exhibit almost no sexual dimorphism  from Eg6. For Eg1, adult specimens of both sexes were available for examination; for Eg2 only juveniles (max. size 35 mm); for Eg3, one adult male and a few juveniles see . For Eg4ism e.g. . Allometism e.g. . No diffE. gryllus on the basis of the sequences available for all specimens (16S rDNA), five of which were also corroborated by the results of the analyses on the three-gene dataset. Analyses based on mitochondrial markers revealed similar clades as those focusing on nuclear data, showing that the results are not an artifact of gene tree – species tree discordance. For all three genes, every clade but Eg4 and Eg5 satisfied the ‘4×’ criterion We identified nine lineages within For COI, a clear barcoding gap could be observed. The level of interclade divergences was found to be in concordance with interspecific divergences reported for lysianassoid amphipods , for which no specimens were available for examination.The current taxonomy of many deep-sea groups is conservative E. gryllus separated by distances up to 16,750 km were characterized by low genetic divergences (COI: 0.8–2.0%) sharing a single 16S haplotype. Gene flow between Antarctic and Arctic populations might be facilitated by passive transport with the thermohaline circulation, i.e. the Antarctic Bottom Water (AABW), originating in the Weddell Sea and covering much of the world ocean floor The bathyal clade Eg1 comprised specimens from both Arctic and Antarctic regions, sampled from 839 to 2,743 m depth. Recently, the Census of Marine Life counted at least 235 organisms  with a bipolar distribution. However, identification of these bipolar species has been exclusively based on morphological characters and might be biased due to morphological convergences or a (perceived) lack of distinctive characters E. gryllus sensu lato did not seem restricted due to geological features, since genetic homogeneity was observed between abyssal populations in the Argentine Basin and around the Antarctic Peninsula as well as between populations in the northwest and northeast Atlantic, across the American Antarctic Ridge and the Mid-Atlantic Ridge, respectively.Hydrography might also play an important role in connectivity between abyssal populations of clade Eg3 for which identical 16S haplotypes were found (i) throughout the entire North Atlantic Ocean, (ii) in the Argentine Basin and around the Antarctic Peninsula and (iii) at two localities in the Central North Pacific. In this case, the Antarctic Circumpolar Current combined with the northbound movements of the AABW might be responsible for dispersal between these basins. Future population genetic studies based on fast-evolving nuclear markers will allow us to test these hypotheses. Oppositely to hydrographic features, topography might also play a role in population connectivity of deep-sea organisms since undersea mountain chains, rises and continents can act as barriers for dispersal E. gryllus seem to be associated with depth. It has been shown previously that small bathymetric changes appear much more significant for promoting population differentiation than geographic distance in a variety of animal groups: corals In contrast to these cases of genetic homogeneity observed over enormous geographic distances, high genetic divergences could be detected over small bathymetric distances. Specimens sampled at 3,070 m near the Antarctic Peninsula (Ant-a) were separated from specimens from the same region (Ant-b) but sampled at shallower depths  by genetic distances in the range of interspecific divergences. This break around 3,000 m also appeared in the trees and haplotype networks, since there was neither cluster nor network that would unite specimens from both above and below this limit. Accordingly, environmental gradients determining the distribution of ParalicellaStephonyx biscayensis, is consistent with its bathymetric and geographic distribution E. gryllus, a genetic separation appeared between 2,743  and 3,070 m depth. For the studied specimens of E. gryllus by France and Kocher Deminucula atacellana between 3,000 and 3,300 m depth E. gryllus can easily tolerate pressure differences since specimens are easily maintained alive for longer periods when retrieved from depths around 2,000 m . Furthermore, this species is known to accomplish ontogenetic migrations throughout the water column, with adult individuals moving into higher water layers, adopting a pelagic life-style (e.g. E. gryllus is able to migrate vertically through water column without ill effects, our results suggest its reproduction to be limited to a more confined depth range.Vertical partitioning of closely related species has also been observed in other deep-sea benthic yle e.g. . Hence, E. gryllus, as more clades were observed above 3,200 m, than below, and the abyssal lineage was assumed to be widespread More genetic lineages were encountered in abyssal than in bathyal regions: two species-level clades could be distinguished above 3,000 m and seven species-level clades below 3,000 m. This finding is in contrast with the depth-differentiation hypothesis, stating that genetic differentiation between populations decreases with depth (reviewed in Eurythenes gryllus is the most intensively studied taxon of all deep-sea amphipods throughout the world ocean, genetic and morphological differences are still overlooked. Genetic evidence suggests that E. gryllus might represent at least nine species-level lineages, with distinct, partly overlapping, geographical ranges. However, bathymetric distributions seem to be confined to depths either above or below 3,000 m. Two bathyal and two abyssal species-level clades showed a widespread distribution, while five other abyssal species-level clades seemed restricted to a single ocean region. This challenges the general assumption that the abyss has a more limited potential for speciation events to occur due to its homogeneous environment and lack of obvious isolating barriers. If such unexpectedly high species diversity is observed for a giant and highly mobile amphipod, we predict the abyss to be much more complex and more likely to harbor an important hidden invertebrate diversity than previously assumed. Molecular studies supporting the depth-differentiation hypothesis are scarce and often based on a limited sampling. Extending the sampling coverage gave contrasting results to previous assumptions. These biases due to sampling effort clearly highlight the difficult nature of deep-sea research. In addition, deep-sea organisms are known to be patchily distributed (e.g. Although ted e.g. , which i