The approaches were mainly synchronic comparisons of phenotypically divergent populations, substituting spatial contrasts in temperature or CO2 environments for temporal changes, or in assessments of adaptive genetic diversity within populations for traits important under global change. The available literature is biased towards gastropods, crustaceans, cnidarians and macroalgae. Focal traits were mostly environmental tolerances, which correspond to phenotypic buffering, a plasticity type that maintains a functional phenotype despite external disturbance. Almost all studies address coastal species that are already today exposed to fluctuations in temperature, pH and oxygen levels. Recommendations for future research include (i) initiation and analyses of observational and experimental temporal studies encompassing diverse phenotypic traits  (ii) quantification of nongenetic trans-generational effects along with components of additive genetic variance (iii) adaptive changes in microbe–host associations under the holobiont model in response to global change (iv) evolution of plasticity patterns under increasingly fluctuating environments and extreme conditions and (v) joint consideration of demography and evolutionary adaptation in evolutionary rescue approaches.I summarize marine studies on plastic versus adaptive responses to global change. Due to the lack of time series, this review focuses largely on the  The ocean is by far the largest habitat on planet Earth. Even larger is our level of scientific ignorance with respect to basic knowledge on its biodiversity. While the recently completed census of marine life compiled a list of 240 000 metazoan species known to science, three to nine times more species still await discovery and description, depending on the extrapolation approach used  or reciprocal transplant approaches. In such studies, the phenotypically plastic component of phenotypic variance is usually not directly estimated, but contained within the error variance. Indirect evidence is also available from assessments of relevant within-population genetic diversity, for example in tolerance traits in the face of warming or ocean acidification stress, which may then be combined with population genetic projections on adaptation rates  and Ne is <1, then, random processes (genetic drift) will constrain adaptive responses via selection. Population size has been invoked to be one key variable for the possibility of evolutionary rescue (ER) of populations under changing environments, either by determining the amount of quantitative genetic variation responsive to selection, or indirectly via inbreeding effects  periods or entire regions in both open ocean and coastal areas (Diaz and Rosenberg 2) will enhance the growth of marine plants   Fig. , the reant) Fig. . The latnt) Fig. . In contThe two plasticity types are associated with different modes of selection by global change. Classical plasticity is most relevant under selection for enhanced opportunity  are broadly consistent with the thermal niche of a population, that is, individuals from colder locations indicated heat stress at lower temperatures by expressing hsp genes in marine invertebrates and seagrasses (Osovitz and Hofmann Strongylocentrotus purpuratus) , ideally under at least two levels of that factor to unravel G × E (genotype × environment) interactions  limitation and enhances growth , adaptive phenotypic divergence at the population level was mainly studied with respect to temperature regimes , either Increasing mean temperature predicted for many regions will strongly interact with genetically based seasonality patterns that are probably highly adaptive both within and among species. Warmer waters may enable local algal populations to grow longer time periods when conditions become more favourable, typically at colder sections of their current distribution range Eggert . HoweverZostera marina (eelgrass), a series of common garden experiments have revealed some evidence for thermal adaptation of southern versus northern populations in terms of their photophysiology , gastropod molluscs and copepods (Crustacea). Most studies used controlled laboratory common garden designs under space-for-time substitution approach, manipulating either a range of temperatures including stressful values, or only temperature as stressor . About h, Willet  found thLabidocera aestiva), the production of dormant eggs was population specific, suggesting local adaptation of developmental mode to the length of the growing season, which is covarying with temperature  from populations diverging in the pH environment their parents experience along the Pacific coastline, owing to different upwelling regimes along with oxygen deficiency and naturally occurring pH drops. The maintenance of larval size was related to experimental ocean acidification stress in a way consistent with local adaptation to naturally occurring pH value decreases.There are far fewer studies addressing adaptation to ocean acidification using a synchronic approach. Using the well-defined COIn synchronic approaches, it is mandatory to erase environmental effects that persist within or even across generations to correctly infer evolutionary adaptation. Unfortunately, even long-term acclimation within generations may be insufficient to erase irreversible environmental effects. For example, early ontogenetic effects on muscle morphology and swimming performance in zebrafish were found to be unaffected by subsequent acclimation of adults to different thermal regimes  because larvae of the latter possessed lower levels of additive genetic variance. The above study only addressed very early larval stages and needs to be extended to later life stages. Other such recent examples include the variation in larval tolerance in a sea urchin to the combined effects of warming and ocean acidification  in many areas of the world Rowan . The surTigriopsis spp. from northern locations failed to adapt to temperature stress in 10 generations of adaptation. Note that Tigriopsis is a rather atypical marine invertebrate occurring in exceptionally small, isolated populations. Hence, one likely explanation for the observed evolutionary constraint is the lack of standing genetic variation as a consequence of small Ne and associated genetic drift  corals, where Symbiodinium photosynthesis provides the host–symbiont association with >90% of its nutrition. Many more examples are appearing in other invertebrates and plants, such as in sponges , for example in photoperiodic cues for sporulation or flowering dates , in activity or migration patterns or in seasonal energy allocation patterns. It is also clear that even if initiated now, such time series would start to become instructive only much later. Some preserved specimen collections may be instructive to at least determine morphological shifts in, for example body size and form. An interesting alternative over monitoring programmes may be time series of revived genotypes obtained from resting stages stored in laminated sediments, for example from copepod resting eggs  changes at the molecular level  were up to 50% smaller when accounting for adaptive evolution in a model considering measurements of additive genetic variance for size (as proxy for fitness) and predicting the rate at which a suboptimal phenotype returns to its optimal state by stabilizing selection. Note that approaches using h2 and selection differentials are a useful first step, but they have their inherent shortcomings. For example, due to trait correlations, the erosion of genetic variance under strong directional selection and fluctuating selection regimes, their predictions are often not very accurate  could hence be a precursor of mean trait changes. Alternatively, direct evolution of increasing plasticity, in the sense of steeper reaction norm slopes, is also possible and may be favoured by enhanced environmental variability predicted under global change  evolution, based on genetic changes in wild populations. Owing to the lack of time series in marine populations other than abundance and distribution, I have largely explored the scattered evidence for the potential of adaptive evolution in the wild. Clearly, it will be impossible to study the entire taxonomic diversity of marine animals and plants, but a more systematic study of major life-history types, population sizes, habitat types  and migration capacities is highly warranted.


Dipteryx alata . We then obtained a series of genetic parameters for the species after generating extinctions in areas of low future habitat suitability.Many species are expected to suffer a strong shift in geographic ranges due to climate changes in the next fifty years, depending on their ecological tolerance and current demographical parameters, which were in turn shaped by their evolutionary history. These shifts may also cause a change in genetic population structure and variability, because local extinctions or reduction in fitness are not expected to be random in geographical space. Here we used an ensemble forecast approach of Species Distribution Modeling  to derive current and future geographic distribution of the Neotropical tree D. alata throughout the Brazilian Cerrado, which were recorded in a grid with 6240 cells of 0.5o of latitude/longitude covering South America. These occurrences were modeled as a function of eight climatic variables (WORLDCLIM), for the current time and projected into2050,for three different Global Circulation Models  [We obtained a total of 448 occurrences of  HadCm3) ,2.Occurrences were modeled using six different SDM techniques . MethodsD. alata consisted in microsatellites markers analyzed for 25 widely distributed local populations, encompassing species’ geographical range. A total of 644 individual trees were genotyped for eight microsatellite loci. Genetic parameters were used to estimate the total amount of polymorphism and genetic diversity currently found in D. alata. We estimated the number of alleles per locus, genetic diversity (expected heterozygosity under Hardy–Weinberg equilibrium), and the expected heterozygosity under mutation-drift equilibrium. Moreover, we recalculated these parameters by assuming that, under climate change, there will be a displacement of climatically suitable areas for the species and, consequently, that only populations found in regions above certain levels of suitability in the future will persist.Genetic data for D. alata will shift its geographic range and climatically suitable areas from Central towards Southeastern Brazil . Because of the wide range, currently and in the future, some of the local populations with highest genetic diversity will potentially remain in highly suitable areas in the future, even using very conservative thresholds of 80%. However, it is important to highlight that coalescence analyses and the expected heterozygosity under mutation-drift equilibrium suggest strong population bottlenecks for the species in the recent past, which were corroborated by hind cast projections of the SDMs using paleoclimatic data. This explains the low level of genetic variability in D. alata, despite its wide geographic range.The wide current range and ecological tolerance of D. alata. Even so, it is important to realize that these results are mainly due to the relatively low current genetic variability of the species, probably associated with recent population bottlenecks. In this case, climatic shifts can have more serious adaptive consequences and further investigations are necessary to avoid a false indicative of high probability of persistence of this species based on a low relative level of loss of genetic diversity.Thus, despite the shift in geographic range size and climatically suitable areas towards Southeastern Brazil and the expected downward shift in the genetic parameters, the analyses performed here do not show a strong loss of genetic diversity in


Centroscymnus crepidater is a medium-sized benthopelagic species that exhibits a circumglobal distribution occurring both in the Atlantic and Indo-Pacific Oceans. Contrary to the wealth of phylogeographic studies focused on coastal sharks, the genetic structure of bathyal species remains largely unexplored. We used a fragment of the mitochondrial DNA control region, and microsatellite data, to examine genetic structure in C. crepidater collected from the Atlantic Ocean, Tasman Sea, and southern Pacific Ocean (Chatham Rise). Two deeply divergent (3.1%) mtDNA clades were recovered, with one clade including both Atlantic and Pacific specimens, and the other composed of Atlantic samples with a single specimen from the Pacific (Chatham Rise). Bayesian analyses estimated this splitting in the Miocene at about 15 million years ago. The ancestral C. crepidater lineage was probably widely distributed in the Atlantic and Indo-Pacific Oceans. The oceanic cooling observed during the Miocene due to an Antarctic glaciation and the Tethys closure caused changes in environmental conditions that presumably restricted gene flow between basins. Fluctuations in food resources in the Southern Ocean might have promoted the dispersal of C. crepidater throughout the northern Atlantic where habitat conditions were more suitable during the Miocene. The significant genetic structure revealed by microsatellite data suggests the existence of present-day barriers to gene flow between the Atlantic and Pacific populations most likely due to the influence of the Agulhas Current retroflection on prey movements.Unravelling the genetic structure and phylogeographic patterns of deep-sea sharks is particularly challenging given the inherent difficulty in obtaining samples. The deep-sea shark  The paucity of data on deep-sea sharks due to the intrinsic difficulty in obtaining samples, have hampered our understanding of the genetic structure of the species inhabiting these remote environments. Nevertheless, a number of expectations based on the prior knowledge of this group of species can be formulated. Dispersal in elasmobranchs is mostly determined by the swimming ability of juveniles and adults, as they have no pelagic larvae. Vagility increases with body size and tends to be higher in pelagic or benthopelagic oceanic species, whereas benthic and coastal species often exhibit low dispersal abilities Large oceanic elasmobranchs are expected to exhibit low levels of genetic differentiation across vast stretches of the open ocean whereas smaller benthic species may display stronger genetic differentiation. While there is a relatively large number of phylogeographic studies focused on coastal sharks An increasing number of studies in elasmobranchs reported genetic discontinuities within ocean basins, revealing the existence of barriers to gene flow Paleoceanographic changes including the opening and closure of corridors connecting marine basins triggered major climate fluctuations as well as faunal turnover. There is increasing evidence that Tethys final closure at 14 myr Centroscymnus crepidater  is a deep-sea shark with an estimated age of 20 years for female maturity C. crepidater exhibits a worldwide distribution occurring on the continental and insular slopes of the eastern Atlantic (Iceland to southern Africa), Indian Ocean (Aldabra Islands and India) and Pacific  The long-nosed velvet dogfish C. crepidater belongs to the family Somniosidae, which the paleontological record dates back to the Campanian [83.5−70.6 myr] Centroscymnus are from the Iseyama Formation, central Japan Centroscymnus dates back to the Middle Miocene, and considering its broad distribution with populations occurring both in the Atlantic and Indo-Pacific basins, this tectonic event might have shaped the genetic structure of this species.C. crepidater collected in the Atlantic and southern Pacific Oceans. We further explore phylogenetic patterns using a portion of the mtDNA cytochrome oxidase subunit I (COI) gene. Genetic diversity and connectivity between Atlantic and Pacific populations was examined with seven microsatellite loci. The combined use of mtDNA and microsatellite data allows inferring past evolutionary events C. crepidater reflects any female philopatric behaviour, and to explore if the Tethys closure triggered lineage-splitting events within this species. Given that mtDNA is maternally inherited, it would allow assessing female philopatry. As a result of the Tethys vicariant event, the predicted phylogenetic pattern would be a sister-clade relationship between lineages from the eastern Atlantic and Indo-West Pacific basins Here, we used an 868 bp-fragment of the mitochondrial DNA (mtDNA) control region (CR) to analyse genetic structure in C. crepidater  yielded a total of 70 variable sites and 45 haplotypes  of plotypes . Additioh = 0.886±0.029 and π = 0.0037±0.00038 . Corrected net sequence divergence between clades I and II is 3.1%±0.61%, with an average sequence divergence  between individuals of 1.0%±0.17%. The pairwise estimates of D were only significant between the Azores and the Tasman Sea. Between sites, no pairwise Φst were significant .The haplotype network based on CR sequence data also shows two well-defined clades (hereafter clades I and II) separated by 24 mutational steps . Clade I±0.00038 . Clade Inificant . The indResults of AMOVA between the two oceans (Atlantic and Pacific) using mtDNA sequence data indicate absence of genetic structure, with most of the variation allocated within sites .P<0.001).Mismatch analysis for all the CR mtDNA sequences displays a bimodal distribution with two well-separated peaks . The firC. crepidater from seven Atlantic and two Pacific locations were genotyped for seven microsatellite loci. Gene diversity varied between 0.233 to 0.829 (mean = 0.485) and the total number of alleles per locus varied between 3 to 11 (mean = 7.4)  and non-significant Fis (P>0.132). If only neutral loci were taken into account both values were not significant after correction .A total of 160 individuals of n = 7.4) . The plon = 7.4) . AccordiThe analytical approaches to detect genetic structure were congruent in returning absence of structure within the Atlantic samples but significant differentiation between Atlantic and Pacific. Visual inspection of the scatterplots of the first two principal components of discriminant analysis (representing 29.4% and 8.9%)  showed tayesass showed that contemporary migration between ocean basins is occasional and the direction is predominantly from the Pacific, with 3.8% of individuals migrating into the Atlantic, whereas only 1.9% of the migration occurs in the opposite direction.Results from Beneland analyses recovered three major clusters corresponding to the following locations: (1) Pacific, (2) Atlantic, and (3) Great Meteor Bank  without a discernible geographic pattern. In contrast, microsatellite data indicated a strong genetic differentiation between the Atlantic and Pacific Oceans.Phylogenetic analyses based on mtDNA recovered two deeply divergent clades (24 mutations apart) with an origin dating back to the Miocene. These genetic clades have different abundances  and are equally distributed between ocean basins . Second, the level of interspecific divergence in sharks is generally higher than in bony fishes  is similar to the value found in other shark species  and Indo-West Pacific (another clade) populations. Instead, ML trees  close to the seabed C. crepidater throughout the northern Atlantic where habitat conditions were more suitable during the Miocene C. crepidater (It is not expected that a marked drop in the deep-water temperature could significantly affect a widespread species occupying a wide range of latitudes such as epidater  further Thunnus obesus) C. crepidater. One bigeye tuna clade was ubiquitously composed of Atlantic and Indo-Pacific samples while a second clade occurred almost entirely in the Atlantic. The lower water temperatures during the peak of glacial periods were proposed to restrict migration between ocean basins promoting divergence Two deeply divergent mtDNA clades were described in the bigeye tuna , and it is not unlikely that specimens of C. coelolepis belonging to the putative second clade have not been sampled.The reticulated shape of clade I depicted in the network analysis  suggestsIt is difficult to ascertain the origin of a particular phylogeographic outcome because different biogeographical or stochastic events can result in similar population histories C. crepidater data showed no significant differentiation among Atlantic locations and low levels of gene flow between the Atlantic and Pacific Oceans  or in the presence of oceanic barriers C. crepidater present-day movements by creating a filter to the displacement of its prey between ocean basins. Coastal organisms such as intertidal mussels (Perna perna), exhibit distinct lineages around the South Africa coastline, which were attributed to a filter role of the Agulhas Current retroflection At the interface between the Atlantic and the Indian Oceans, one of the main oceanographic current is the Agulhas Current (southward flowing) and its retroflection formed by the turning back into the Indian Ocean C. crepidater. Moreover, results based either in mtDNA or microsatellites showed absence of genetic or geographic structure across putative preferential areas of homing behaviour such as submarine banks  that are usually used as breeding sites Recent studies on sharks ascribed genetic differentiation to female philopatric behaviour  aboard longliners. Permissions to join the commercial vessel crews were granted by the local Governments of Madeira and Azores for scientific purposes only. MAR and MET sampling locations are located in high seas, which means in legal terms, in areas beyond national jurisdiction, and no further permits are required. In regards of the killing, the majority of deep-sea sharks by the time they reach deck are already dead. For the specimens that were showing signs of life, they were released after a very quick and delicate handling to obtain info on length, weight and sex. Muscle tissue samples were collected from dead specimens.All samples from the Azores, Madeira, Mid-Atlantic Ridge (MAR), and Great Meteor Bank (MET) used in this study originated as by-catch from commercial fisheries and were collected by personnel at the Department of Oceanography and Fisheries from the Azores  or oreo dories . The sharks would have been brought to surface (from depths of up to 1200 meters) dead amongst the large catch of either Orange Roughy and/or oreo dories. The tissues samples were taken from dead specimens. The specimens were later examined for various biological parameters  by researchers from our divisional shark research team. Samples from the Chatham Rise (southern Pacific Ocean) were obtained through the Bavarian State Collection of Zoology DNA Bank. Centroscymnus crepidater is NOT listed in the IUCN list of endangered/vulnerable/threatened species.We obtained permits from CITES to import tissue samples from the Tasman Sea through the CSIRO - Marine and Atmospheric Research Institute . All 22 samples of Centroscymnus crepidater) were collected between 2003–2009 from seven locations in the Atlantic Ocean , 0.2 mM of each dNTP, 0.2 µM of each primer, 1 µl of template DNA, and Taq DNA polymerase . The following program was used for the PCR amplification: one cycle of 1 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 50°C (COI) or 52°C (CR), and 60 s (COI) or 70 s (CR) at 68°C, and finally, one cycle of 5 min at 68°C. PCR products were purified with an ethanol/sodium acetate precipitation, and directly sequenced using the corresponding PCR primers. Samples were sequenced in an automated DNA sequencer (ABI PRISM 3700) using the BigDye Deoxy Terminator cycle-sequencing kit (Applied Biosystems) following manufacture’s instructions.All PCR amplifications were conducted in 25 µl reactions containing 1X PCR buffer (C. crepidater (132 specimens from the Atlantic Ocean and 28 from the Pacific Ocean). PCR amplifications were carried out using Qiagen Multiplex PCR kit in a final volume of 10 µl containing 5 µl of Multiplex Kit Buffer 2× and 2.5 ng/µl of genomic DNA. Final primers’ concentrations and fluorescent labels (Applied Biosystems) were as follows: Ccrep02 [NED] 0.45 µM, Ccrep11 [PET] 0.15 µM, Ccrep12 [6-FAM] 0.3 µM, Ccrep13 [6-FAM] 0.2 µM, Ccrep23 [VIC] 0.25 µM, Ccrep27 [VIC] 0.1 µM and Ccrep34 [NED] 0.15 µM. Amplification started at 95°C for 15 min, followed by 35 cycles of 45 sec at 94°C, 45 sec at 59°C, 45 sec at 72°C, and a final extension step of 45 min at 72°C. PCR products were run alongside a GS600LIZ size standard (Applied Biosystems) in an ABI 3130xl Genetic Analyzer and alleles were scored using the program Genemapper 4.0 (Applied Biosystems).Seven species-specific microsatellites  multiplexed in a single PCR reaction C. crepidater: 66 from the Atlantic Ocean; 21 from the Tasman Sea; five from the Chatham Rise) were aligned with ClustalX Centroscymnus owstonii  and one sequence of Centroscymnus coelolepis  as an outgroup, given the close phylogenetic relationship between these species C. crepidater  were also aligned with ClustalX. In the ML analysis based on the mtDNA COI data set, we used Centroscymnus coelolepis as outgroup (DQ108219).CR mtDNA sequences  and nucleotide (π) diversities rlequin v3.5 DD and its statistical significance values, obtained by 1000 permutations st (mtDNA) values generated in Arlequin to readily enable comparison with other studies given the widespread use of this statistic. Genetic diversity within and among populations was estimated with an analysis of molecular variance , and within each ocean basin. The significance of all Φ-statistics was tested by 10,000 random permutations of sequences among populations.Haplotype . The sudden expansion sum of squared deviation (deviation of simulated from observed – SSD) was calculated and tested against that expected under the sudden expansion model. We tested for deviations from neutral expectations using Fu's Fs2naSP and significance was evaluated by comparing the observed statistics to a distribution of values generated with 5000 coalescent simulations.Signatures of changes in population size can be detected in the pattern of pairwise differences among mitochondrial sequences enoDive v2.0b22 O); (3) expected heterozygosity (HE); (4) population gene diversity (HS), and (5) total corrected gene diversity (H’T). Allelic richness (AR), a sample-size independent measure of the number of alleles, was estimated using Fstat v2.9.3.2 rlequin using microsatellite data. We used the StandArich_v1.00 http://www.ccmar.ualg.pt/maree/software.php?soft=sarich).We used genotype and allele frequencies of the microsatellite loci to obtain standard estimates of genetic diversity within and between sample sites. For each microsatellite locus and each location, genetic diversity was assessed with Gst-outlier approach ositanST’ option We tested for signs of positive and balancing selection using the Fisis statistics from an AMOVA enoDive. Differentiation among samples was estimated using Jost’s Dtics v. 0.8–5 D values was tested using 1,000 bootstraps. For completeness, we also provide Fst (microsatellites) values and their statistic significance to readily enable comparison with other studies given the widespread use of this statistic.Deviations from the Hardy–Weinberg Equilibrium (HWE) were assessed with the Gdegenetfind.clust was employed to investigate genetic structure by running successive k-means, using the Bayesian Information Criterion (BIC) and 107 iterations. The optimal k is usually associated with the lowest BIC value. In cases of continuously decreasing BIC, the optimal k could be selected by visually investigating the rate of decrease Discriminate Analysis of Principle Components (DAPC) value package When appropriate, significance levels of all multiple statistical tests were corrected using the false discovery rate (FDR) approach ayesass6 MCMC iterations and a burn-in of 2×106 steps. The data was run three times to check for consistency of results, and their values were averaged.Contemporary direction and rate of migration was estimated by a Bayesian method based on multi-locus genotypes implemented in Beneland version 2.0 eneland is able to detect subtle geographic structure by combining geographic data into the analysis as a weak prior. This follows the assumption that each population exhibits some degree of spatial structure; however, individuals can belong to multiple populations for posterior likelihood testing Spatial structure was assessed with a Bayesian clustering algorithm to determine the most probable number of genetic clusters present within the dataset without prior knowledge of the individuals’ origin. This method of population assignment was analyzed using the R package Gollapse version 1.2 C. crepidater, we used Multidivtimehyml as a starting phylogeny. Two calibration points were set according to the fossil record. The first calibration was based on the minimum age of the family Somniosidae Centroscymnus from the Middle Miocene of Japan rtrate was set to 0.014 (mean of the prior distribution for the rate at the ingroup root node) and the standard deviation was set to its maximum value . The MCMC method was employed to approximate both prior and posterior distributions Prior to the analysis, identical haplotypes from the mtDNA CR data set were collapsed with the program CMaterial S1Phylogenetic relationships within Centroscymnus crepidater based on a maximum likelihood analysis of an 868 bp fragment of the mitochondrial control region. Numbers above and below nodes correspond to ML bootstrap proportions and Bayesian posterior probabilities, respectively.(EPS)Click here for additional data file.Material S2Phylogenetic relationships within Centroscymnus crepidater based on a maximum likelihood analysis of a 655 bp fragment of the mitochondrial cytochrome oxidase subunit I. Numbers above and below nodes correspond to ML bootstrap proportions and Bayesian posterior probabilities, respectively.(EPS)Click here for additional data file.Material S3Allele distributions. Distribution of the alleles found for each locus at the different sites sampled. Note that not all alleles are represented numerically underneath the respective plot. However, the allelic distribution is represented in the graphs for all alleles.(TIF)Click here for additional data file.Material S4eneland analysis.Posterior density distribution of the number of clusters estimated from GA. Including the Great Meteor Bank sample. B. Excluding the Great Meteor Bank sample.(TIF)Click here for additional data file.


Napier grass is an important forage for smallholder dairy farms. However, there has been a comparatively low effort to improve Napier grass. It is necessary to strengthen forage breeding programs for development of cultivars with superior traits like. With a high rich gene pool; correct identification of Napier grass accessions is a prerequisite because the existing germplasm information is scanty and cannot be relied upon for crop improvement. Thus the genetic assessment of various Napier grass accessions from the Eastern Africa region is important for correct cultivar identification in order to fully exploit them in crop improvement strategies. Fst value obtained was moderately significant (Fst = 0.1688). Neighbour-joining analysis gave two distinct clusters which did not reflect geographical locations. Analysis of molecular variance showed all variance components to be highly significant (P < 0.001), indicating more variation within (91 %) than between populations (9 %). Results suggested moderate genetic differentiation among Napier grass populations sampled, which could imply a high germplasm exchange within the region. The AFLP markers used in this study efficiently discriminate among cultivars and could be useful in identification and germplasm conservation.Napier grass is an important forage crop for dairy production in the tropics; as such, its existing genetic diversity needs to be assessed for conservation. The current study assessed the genetic variation of Napier grass collections from selected regions in Eastern Africa and the International Livestock Research Institute Forage Germplasm-Ethiopia. The diversity of 281 cultivars was investigated using five selective amplified fragment length polymorphism (AFLP) markers and classical population genetic parameters analysed using various software. The number of bands generated was 216 with fragments per primer set ranging from 50 to 115. Mean percentage polymorphic loci was 63.40. Genetic diversity coefficients based on Nei's genetic diversity ranged from 0.0783 to 0.2142 and Shannon's information index ranged from 0.1293 to 0.3445. The  Pennisetum purpureum) is a perennial grass grown widely as a fodder crop and feed for the cut-and-carry zero-grazing dairy systems  restriction fragment length polymorphisms (RFLPs) are time consuming and involve expensive radioactive materials , (iii) iThis study assessed the genetic variation between and within Napier grass collections comprising 281 accessions from selected regions in Eastern Africa . Implications of the findings for germplasm identification, breeding and conservation are discussed.Cultivars were collected from selected regions in Kenya, Uganda and Tanzania between 2009 and 2010. Also included was a collection from the International Livestock Research Institute Forage Germplasm (ILRI-FG) which comprised cultivars from other parts of Africa, USA and accessions of unknown origin. The ILRI-FG was established in 1982 and has been maintained without external infusion  ) was added and the samples homogenized for 1 min by re-grinding in a GenoGrinder. Samples were incubated in a water bath at 65 °C for an hour with continuous shaking and mixing by inversion of strip tubes (every 15 min). Tubes were then removed from the water bath and left to cool at room temperature for 10 min, followed by centrifugation at 4000 rpm for 20 min. The aqueous layer was transferred into fresh strip tubes. Chloroform : isoamyl alcohol  was added and the contents mixed by gently inverting the tubes 10 times. Centrifugation was carried out at 4000 rpm for 20 min and the aqueous (top) layer transferred into a fresh strip tube. The above step was repeated twice and the aqueous layer transferred into fresh strip tubes. Ice-cold absolute ethanol : sodium acetate  was added gently and mixed by inversion, and a sample incubated at −20 °C for 45 min. DNA was pelleted by centrifugation at 4000 rpm for 20 min. The supernatant was removed; the pellet was washed with 500 µL of 70 % ethanol and left to stand for 5 min, then washed twice. The DNA pellet was air dried for 20 min, then dissolved in 100 µL of low-salt TE buffer . To free the DNA of RNA, 5 µL of ribonuclease A (RNase A), 10 mg/mL, were added and incubated at 37 °C for 30 min. DNA was re-precipitated by adding 200 µL of cold absolute ethanol and incubated at −20 °C for 30 min. DNA was pelleted by centrifuging at 4000 rpm for 20 min and washed with 70 % ethanol twice. The pellet was air dried for 30–60 min and resuspended in 100 µL of low-salt TE buffer.Genomic DNA was extracted following the cetyltrimethylammonium bromide (CTAB) method  with somxl Genetic Analyzer (Applied Biosystems). Amplified fragment length polymorphism peaks from the Genetic Analyzer were sized and alleles scored with GeneMapper version 4.1 . Scored results were exported to an Excel matrix with values 1  or 0 .The AFLP protocol described by F statistics, gene diversity over loci, proportion of polymorphic loci, Shannon index and gene frequency —for diversity among and within populations—were determined using GenAlEx software . PopGen3requency . In addirequency . To showrequency . About 2From the initial 64 primers screened, 22 were efficient at the inter-population level and sufficiently polymorphic to discriminate clones within populations. Intra-population polymorphism obtained with the selected primers gave alleles unique to each individual from the same population. Five primers that generated electropherograms with high relative fluorescent units/(peaks) without background noise were further selected for genotyping. Amplified fragment length polymorphism fragments ranged from 50 to 500 base pairs. Polymorphic bands ranged from 50 to 115 with an average of 43 bands per primer from a total of 216 bands generated, accounting for 64.80 % of polymorphic loci observed. The percentage of polymorphic fragments within sub-populations ranged from 32.5 for the National Agricultural Research Organization (NARO) to 89.4 % for Mumias. Following the same order, the genetic diversity coefficients based on Nei's genetic diversity ranged from 0.0783 to 0.2142 and Shannon's information index ranged from 0.1293 to 0.3445 (Table The genetic distances between/among 21 Napier grass sub-populations (regions within countries) and four populations  were subjected to hierarchical clustering by UPGMA Table . This yiP < 0.001) with more variation within (91 %) than between populations (9 %) (Table P < 0.001). On the other hand, variation among population showed the Kenyan population as having the least variation (3 %) while the most variation was among the Ugandan population (17 %).Variance components obtained by AMOVA were highly significant (%) Table . Similarn = 4x = 28) and triploid and hexaploid hybrids occur between it and pearl millet , which are in a radius of ∼60 km. Similarly, this applies to ILRI-FG, Tanzania and Uganda populations, which had moderate genetic distances. The ‘Tanzania’ sub-population, from ILRI-FG, clustered with Tanzanian populations, Lushoto, Tarime and Meru, indicating ancestry from Tanzania. The sub-ILRI-FG germplasm is diverse with most originating from Zimbabwe and the USA; however, their origin could not be ascertained. The USA is physically isolated from Africa, but these individuals were still flanked by the cultivars collected from Africa. This could suggest that they had not yet diversified from their African relatives. The current study was founded on the premise that Napier grass accessions could segregate based on geographical origin and hence would offer a unique genetic resource for breeding programmes in the region. However, the converse appears true as there is moderate diversity among accessions in the region. The clustering suggests genotype overlap and redundancy possibly due to low gene flow. The materials under circulation share ancestry especially since Napier grass is clonally propagated, the users may be sharing planting materials. These findings contradict previous studies that demonstrated clustering of Napier grass accessions based on geographical location (Lolium perenne), meadow fescue (Festuca pratensis), orchard grass (Dactylis glomerata) and Rhodes grass (Chloris gayana) . They cross easily but their hybrids are sterile. The tertiary gene pool comprises true biological species compared with the primary and secondary gene pools  with materials from different agroecological zones of the world. Potential sources of Napier grass diversity are South Africa, Brazil, Puerto Rico, the USA, Australia, China, Pakistan and India (Assessment of the entire The AFLP methodology developed in this study was able to discriminate among the Napier grass accessions and could be useful in screening cultivars.Napier grass smut and stunt resistance ASARECA project 06/RC01-FC-2-02) through the Association for Strengthening Agricultural Research in Eastern and Central Africa (ASARECA).This work was supported by a grant (B.W.W. conducted the lab work, and drafted and revised the manuscript with contributions from co-authors. M.O., F.N.W., J. Harvey and R.A.S. were primarily responsible for supervising the laboratory work and revising the manuscript. A.M. provided technical backstopping in the AFLP methodology and data analysis. M.M. was responsible for the field sampling of the Kenyan cultivars. J.P. coordinated all the activities of the project. J. Hanson conceived the study and obtained funding for it. All authors read and approved the final manuscript.None declared.


Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), is a widely distributed and serious economic pest to corn production in the U.S. Genetic variability of O. nubilalis was studied in 18 sub-populations in the upper Midwestern United States using amplified fragment length polymorphism. The relatively low GST values indicate that more variation exists within populations than between populations. High gene flow (Nm) values were indicated across the entire O. nubilalis population; the lowest degree of gene flow was in the northern samples (Nm = 1.96) and the highest degree of gene flow was in the southern samples (Nm = 2.77). The differences observed in the respective regions (north vs. south) may be explained by the voltinism patterns  of O. nubilalis: southern multivoltine populations have opportunities for multiple matings for the duration of the year, further mix alleles. AMOVA results also indicated that most of the genetic variation was within sub-populations ; less variation (≈ 13%) was detected among populations within each of the three regions as designated for this study. However, the most striking and unexpected result was the low percentage of variation between all groups (≈ 6%), further supporting implications of a high degree of gene flow. These results provide support for current requirements of refugia corn planting in Bt-corn management. These results also indicate that if resistance to Bt were to evolve in O. nubilalis, quick action would be necessary to deter the rapid spread of the gene for resistance.The European corn borer,  Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), was introduced into the United States, and is a widely distributed pest that has proven to be a major biotic constraint for maize development and production. O. nubilalis is known to be polyphagous, attacking many herbaceous plants with stems large enough for the larvae to enter. Lewis  in several plant species  where they can provide control of lepidopteran insect pests such as O. nubilalis. With current demands by producers for Bt crops, there is potential for the development of resistance to plant mediated Bt toxins. It is important to develop better understanding of the insect's genetic structure, genetic variation, and gene flow that can provide the basis for improvement and changes in current management strategies for insect control and resistance management.While pesticides have helped control insect pests of food and fiber, pesticide resistance will occur for any pest control product with high selection pressure that removes susceptible individuals from the population and allows only those individuals that possess resistance genes to reproduce with no other intervention . BiotechO. nubilalis, driven by the need to determine the affects of O. nubilalis injury to crops, particularly corn, Zea mays, to assess economic damage and to develop management strategies  markers. Bourguet et al. , Lymantria dispar  in eight states in the upper Midwest and one location from Louisiana (LA) . Specimeµl 1X TE buffer .DNA was isolated from 10 of the 20 individually frozen insect samples using a modified Black and Duteau (1997) CTAB extraction protocol. Prior to homogenization, the gut was removed from the larvae; and the head, wings, and abdomen were removed from adult insects from Mer Rouge, LA. The procedure was modified with the addition of a chloroform/phenol extraction step prior to ethanol precipitation. After removal of ethanol, the pelleted DNA was dissolved in 50 λ DNA  was performed by loading 1.0 µl of DNA mixed with 1.0 µl tracking dye to a 1% agarose gel that was electrophoresed for 30 min. Ethidium bromide stained gels were then visualized using Advanced Quantifier version 4.0 (www.genomicsolutions.com). DNA samples were diluted with TE buffer if concentrations were >22.5 ng//µl.Quantification to a known concentration of EcoRI primers were used in the polymerase chain reaction.The AFLP procedure was achieved using 3 steps: 1) DNA template preparation; 2) DNA template preamplification and; 3) AFLP Selective amplification. Template preparation and AFLP assays were performed using a modified protocol of Vos et al. (1995). Infrared labeled (IRD-700)  µl of volume was incubated with restriction endonucleases EcoRI and MseI  for 2.5 hrs at 37°C in a total volume of 12.5 µl which contained 1.25 µl of 10X One-Phor-All buffer , 0.125 µl of 10 U/µlMseI enzyme (1.25U/reaction), 0.0625 µl of 20 U/µlEcoRI enzyme (1.25 U/reaction), 0.125 µl of 10 mg/ml BSA  (New England Biolabs), and autoclaved nanopure water to make up the volume to 12.5 µl. The restriction fragments were then incubated for 11.5 hrs at ∼ 25°C with 5 µl ligation mixture containing 0.15 µl of T4 DNA ligase, 0.5 µl of 10X T4 ligase buffer (New England Biolabs), 0.5 µl of 5 pmoles/µlEcoRI adapter, 0.5 µl of 5 pmoles/µl MseI adapter , and 3.35 µl autoclaved nanopure water. After ligation, the template was diluted 8-fold. Double stranded adapters for ligation to the corresponding cut made by the restriction enzyme were prepared by incubating equimolar amounts of both adapter strands for 10 min at 65°C, 10 min at 37°C, 10 min at 25°C and stored at -20°C.Approximately 160 ng of genomic DNA in 7 µl of diluted template from adapted DNA diluted 8-fold with IX TE buffer , 8 µl pre-amp primer mix II , 1.0 µl of 10X PCR buffer containing 15mM MgCl2 and 0.25 µl of 5 U/µl AmpliTaq DNA polymerase (1.25 U/reaction) . The oligonucleotide primers in the pre-amp primer mix II are complementary to the adapter/restriction site with MseI primer containing one selective nucleotide M(N+1) primer and EcoRI primer containing no selective nucleotide E(N+O) primer  using 1.0 ) primer . Productµl containing 2.0 µl of diluted preamplified template, ∼ 5.0 µl of autoclaved nanopure water, 1.2 µl of 10X PCR buffer containing 15 mM MgCl2, and 0.06 µl of 5 U/µl AmpliTaq polymerase (Applied Biosystems), 2.0 µl of MseI primer (M+1+2) (6.7 ng/µl dNTPs) (Life Technologies), and 0.2–0.5 µl of 1.0 pmoles/µl IRD-700 labeled EcoRI primer . Selective amplifications were conducted in a DNA thermal cycler 9600 (Applied Biosystems) using the following Touchdown PCR program: 1 cycle of 30s at 94° C, 30s at 65° C and 1 min at 72° C; 12 cycles of 30s at 94° C, 30s at 65°C,  and 1 min at 72° C; 23 cycles of 30s at 94° C, 30s at 56° C, 1 min at 72° C. After selective amplifications, reactions were stopped by adding 2.5 µl blue stop solution (LI-COR).After screening 24 primer combinations, five primer combinations  were selµl of the sample was electrophoresed through KBplus 6.5% ready-to-use gel matrix (LI-COR), and the infrared fluorescent bands were then detected by a laser scanning system LI-COR Model 4200S-2.Samples were then denatured at 95°C for 3 min and flash cooled on ice immediately before loading the polyacrylamide gel for electrophoresis. One Sizes of AFLP fragments were estimated using an IRD-700 labeled 50–700bp ladder. Fragments were scored using SAGA MX version 3.2 (LI-COR); a software program designed specifically for scoring AFLP data. The data were then converted to numerical data  that allowed the identification of polymorphic markers. There were five gels for each primer pair containing four populations randomly picked for a total of 25 PAGE gels. All gels were viewed for clarity prior to scoring to establish scorable bands at a given fragment size for all populations. All bands consistently recognized by SAGA Generation 2 Software Version 3.2 (LI-COR), both monomorphic and polymorphic, were scored and included in the binary data matrix.Analysis was conducted using DBOOT version 1.1  to asses= HT - HS / HT). Gene flow was estimated from GST values and is expressed as (Nm) = 0.5(1 - GST)/GST /GST .A permutation test  was condAnalysis of molecular variance AMOVA;  was condPrincipal component analysis was used to test genetic isolation between sampled populations using NTSYSpc ver. 2.1 . This twO. nubilalis to illustrate genetic similarity. Bootstrap analysis, using BOOD-P software ver. 3.1 (An Unweighted Pair Group Method with Arithmetic Mean (UPGMA) consensus cluster analysis as outlined by Sneath & Skoal  was condver. 3.1 , was useO. nubilalis produced 164 amplified loci, averaged 33 loci per primer combination, and fragment size ranged from 57–341 bp , values calculated were high from all sub-populations, ranging from 0.2370 at Mead, (NE) to 0.3757 at North Platte, (NE); overall gene diversity averaged 0.3094 . AnalysiO. nubilalis range and agree with results of Coates et al. . There was no correlation for the western samples , northern samples , southern samples , nor for the full data set combined . These results further imply there is not genetic isolation in this portion of s et al. .O. nubilalis range.The AMOVA analysis showed that approximately 81% of the variation in the data set was from genotypic variation within populations . Only 13O. nubilalis revealed four distinct clusters , or they may exist as a continuous population where genetic exchange takes place by geographically proximate individuals and there is some genetic isolation by distance . Local selection pressures and barriers could provide for genetic isolation resulting in a high degree of genetic drift. However, previous studies showed a higher degree of variation within populations than between populations for many insects . However, the most striking and unexpected result was the low percentage of variation among groups, further enhancing the implication of a high degree of gene flow possibly through migration events. The degree of variation within or among populations was variable. For many species, variability may be greater between than within populations  and respective UPGMA clustering may be explained by the voltinism patterns that prevail in those regions. The opportunity for multiple mating during the year in southern regions could result in high genetic variability. Another possible explanation of lower indications of gene flow in the northern region could be that s et al. , there ms et al.  evaluateO. nubilalis after release. They found 37–52% of O. nubilalis males recaptured flew 800 m or more, 8–11% flew 3.2 km or more, while some collections of marked males and females occurred at distances greater than 40 km. However, a very large percentage of the O. nubilalis released were never recaptured (over 99.6%). It is possible that some of these individuals traveled much further than placement of the recapture pheromone traps. Showers et al. (O. nubilalis. Although indirect measures of gene flow are tenuous (O. nubilalis were capable of dispersing at least 32 km/yr, would support the concept of gene flow within a continuous population. Results from these ecological and genetic studies provide support for current refuge requirements relative to the use and management of Bt-corn. Also, these results indicate that if insect resistance was to evolve in O. nubilalis, quick action will need to be taken to defer the spread over large geographic areas.Showers et al.  conductes et al.  reported tenuous  our resu tenuous , 2001 il


Maternal inheritance is an essential point in Arabian horse population genetics and strains classification. The mitochondrial DNA (mtDNA) sequencing is a highly informative tool to investigate maternal lineages. We sequenced the whole mtDNA D-loop of 251 Arabian horses to study the genetic diversity and phylogenetic relationships of Arabian populations and to examine the traditional strain classification system that depends on maternal family lines using native Arabian horses from the Middle East.The variability in the upstream region of the D-loop revealed additional differences among the haplotypes that had identical sequences in the hypervariable region 1 (HVR1). While the American-Arabians showed relatively low diversity, the Syrian population was the most variable and contained a very rare and old haplogroup. The Middle Eastern horses had major genetic contributions to the Western horses and there was no clear pattern of differentiation among all tested populations. Our results also showed that several individuals from different strains shared a single haplotype, and individuals from a single strain were represented in clearly separated haplogroups.The whole mtDNA D-loop sequence was more powerful for analysis of the maternal genetic diversity in the Arabian horses than using just the HVR1. Native populations from the Middle East, such as Syrians, could be suggested as a hot spot of genetic diversity and may help in understanding the evolution history of the Arabian horse breed. Most importantly, there was no evidence that the Arabian horse breed has clear subdivisions depending on the traditional maternal based strain classification system. The fact that the Arabian horse breed is one of the oldest pure breeds and among the most widespread on earth is not disputed . It is tThe advent of mitochondrial DNA (mtDNA) sequencing in population genetics during the 1970s produced a revolutionary change regarding historical, biogeographic and phylogenetic perspectives on intra- and inter-species genetic structure . Single Unlike the protein-coding gene region, the displacement loop hyper-variable region of mtDNA (D-loop), represented by about 1200 bp, is of particular interest because of its high level of sequence variation ,22. The RASANs) based upon dam line: Kahlila, Saklawia, Abiah, Shweemat, and Muanakii. The statistical quantities for the DNA sequences, including number of haplotypes and haplotype diversity and nucleotide diversity, were carried out using DnaSP 5.10.1 [We designed two pairs of primers based upon the horse mtDNA sequence reference X79547 . We alsoP 5.10.1 . The staEquus asinus) mtDNA sequence [NCBI: nc_001788] [Phylogenetic analysis of the haplotypes using a whole D-loop sequence was carried out with the PHYLIP software package  based on_001788]  was used_001788] ,11.http://fluxus-engineering.com). Default settings were applied  [Another approach for phylog enetic analysis was carried out by drawing the median-joining network (MJ network)  in accor, ϵ = 0) , and pre, ϵ = 0) ,16. Each, ϵ = 0) .ST were applied. PCoA of the dissimilarity matrix according to Kimura [ST were done using the Kimura 2-parameter model with 1000 permutations and were carried out with Arlequin 3 [ST results, we followed the suggestion that refers that a value between 0–0.05 indicates little genetic differentiation; a value between 0.05 and 0.15, moderate differentiation; a value between 0.15 and 0.25, great differentiation; and values above 0.25, very great genetic differentiation [To represent the genetic structure and differentiation of tested populations, principal coordinate analysis (PCoA), analysis of molecular variance (AMOVA) and pair-wise Fo Kimura  based ono Kimura ,52. AMOVlequin 3 . For thentiation ,55.We declare that we have no competing interests related to this study.AMK and EGC designed the experiment. AMK collected the Syrian Samples, performed the lab works and data analysis and wrote the manuscript. EGC provided the other tested samples, revised the manuscript and supervised the project. AMK and EGC read and approved the final manuscript.Populations sampling background and sampling permissions.Click here for file


Chaetodon tricinctus) using mt and msatDNA (nuclear microsatellites) to distinguish evolutionary versus contemporary gene flow, estimate self-replenishment and measure genetic diversity among locations at the remote Australian offshore coral reefs of Middleton Reef (MR), Elizabeth Reef (ER), Lord Howe Island (LHI), and Norfolk Island (NI). Mt and msatDNA suggested genetic differentiation of the most peripheral location (NI) from the remaining three locations . Despite high levels of mtDNA gene flow, there is limited msatDNA gene flow with evidence of high levels of self-replenishment (≥76%) at all four locations. Taken together, this suggests prolonged population recovery times following population declines. The peripheral population (NI) is most vulnerable to local extinction due to its relative isolation, extreme levels of self-replenishment (95%), and low contemporary abundance.Extensive ongoing degradation of coral reef habitats worldwide has lead to declines in abundance of coral reef fishes and local extinction of some species. Those most vulnerable are ecological specialists and endemic species. Determining connectivity between locations is vital to understanding recovery and long-term persistence of these species following local extinction. This study explored population connectivity in the ecologically-specialized endemic three-striped butterflyfish ( Coral reef fishes have evolved in a close relationship with coral reef habitats to produce the most diverse vertebrate communities on earth Bellwood . HoweverIn terrestrial habitats, endemic species (particularly on isolated islands) typically have higher rates of extinction and lower genetic diversity Frankham . Coral rTo thoroughly understand gene flow it is important that both evolutionary and contemporary levels of gene flow are determined , by complete sampling across its four geographically isolated locations : Middleton Reef − MR, Elizabeth Reef − ER, Lord Howe Island − LHI, and Norfolk Island − NI.Large data sets of highly polymorphic msatDNA loci (nuclear microsatellites) produced by next generation sequencing , revealed limited contemporary gene flow between ER, MR, and LHI , is typical of butterflyfishes  to determine patterns and levels of gene flow between locations using mtDNA  and msatDNA; (ii) to estimate levels of self-replenishment  and recent migration; and (iii) to measure population genetic diversities at all locations as an indicator of potential resilience of populations to environmental change and extinction.n = 30), ER (n = 31), LHI (n = 26), and NI (n = 21). We used a large number of polymorphic microsatellite loci (n = 20) and sampled all known locations, to compensate for the small sample sizes used in this study  as a model organism. Fin clip samples were obtained from fishes of adult size  either by spearfishing or by anesthetizing fish with clove oil, which were fin clipped in situ and released alive .The main aim of this study was to determine gene flow between and genetic diversity at isolated locations using the endemic three-striped butterflyfish , C. plebius (AF108602.1), and C. bennetti (FJ167686.1) based on the findings of Bellwood et al. (C. tricinctus from the four locations.mtDNA Cytochrome b (cyt b) sequence data were obtained from GenBank for the following three most closely related species which acted as out-groups: d et al. . jModeltd et al.  identifid et al. ,b. This d et al.  to expliC. tricinctus were estimated between or within each of the four locations using MIGRATE-n 2.4.3 , custom-migration models  and a geographic matrix – all with a constant mutation rate. A Log Maximum-Likelihood analysis (Ln ML) selected a migration prior (Fst), custom-migration model (migration model with variable Theta), constant mutation rate with an F84 mutation model, migration rate parameters , and a Bayesian analysis, using a heating search strategy of one long chain that sampled every 20th of 60 k sampled trees and applied a 20 k iteration burn-in. All parameters converged and fell within the 90% CI yielding values for θ and M  per location.mtDNA migration rates and effective population sizes of K, we followed the method suggested by Pritchard et al.  Discriminant Analysis of Principal Components  using MIGRATE-n 2.4.3 as above. We set datatype to Microsatellite (a simple electrophoretic ladder model), migration prior (Fst), custom-migration model (migration model with variable Theta), constant mutation rate with a stepwise mutation, migration rate parameters , and a Bayesian analysis, using a heating search strategy of one long chain that sampled every 20th of 60 k sampled trees and applied a 20 k iteration burn-in. All parameters converged and fell within the 90% CI yielding values for θ and M  per location.Contemporary migration rates and effective population sizes of This study did not sample new butterflyfish recruits in order to determine self-recruitment as in Jones et al. . Howeverh; nucleotide diversity, π) and for msatDNA  were estimated in ARLEQUIN 3.5  <0.5 or high if values of h and π (%) were >0.5  contained exclusively individuals from the peripheral location. The other, Clade 2 (n = 6), comprised of an equal frequency of all three western locations  of all individuals, and 12 of which were unique to single fish only in the sample examined here, nine of which were from the peripheral location . Haplotype and genotype diversities were low (<0.5) within the western region, but high (>0.5) at the peripheral location (gd), in contrast, was high at three of the four locations, ER being the exception. Detailed genetic diversity, AMOVA, summary statistics, pairwise population comparisons, and locus by locus AMOVA can be found in Supporting Information .Two hundred and eighty-three base pairs of mtDNA (cyt b) were resolved for 97 location B. Mt andlocation  and S1. FIS; Heterozygote excess was evident from a negative inbreeding coefficient FIS; ; althougPatterns and levels of gene flow based on an mtDNA AMOVA indicated significant genetic variation (65.77%) within locations, Φst = 0.342  locus by locus analyses corrected for null allele frequency . In contrast, an Excluding Null Alleles (ENA) corrected msatDNA pairwise Fst value showed no significant genetic differentiation between any of the four locations as estimates of genetic differentiation between locations fell within 95% confidence intervals ; thereby identifying four genetically differentiated populations. Similarly, four geographically partitioned populations were identified by STRUCTURE analyses, as the likelihood of the marginal posterior probability distribution was highest when K = 4.DAPC, GeneClass2, and STRUCTURE confirmed the presence of at least three distinct genetic populations corresponding to geographic location. DAPC partitioned location C. Using Migrate-n indicated a few orders of magnitude lower levels of contemporary gene flow between locations when compared to mtDNA gene flow, with M values ranging from 2 to 6 B.Fst) between locations within the western region, both DAPC and STRUCTURE partitioned the data into at least three distinct clusters. Used together, these programs are likely to be better than Fst values  and loci are in linkage equilibrium. Moreover, if the above-mentioned conditions are not met, then accurate estimates of migration rates are obtained only when migration rates are very low (m = 0.01) and genetic differentiation between locations is high  and possibly LHI to ER  and nucleotide diversity (% π) in all three western locations , whilst the peripheral location had four- to eightfold higher h and % π , ER being the exception  sufficient mtDNA gene flow connecting all locations within the western region, but low gene flow and consequent isolation of the peripheral population from the western locations; (ii) low msatDNA gene flow between all locations resulting in populations that are genetically differentiated; (iii) demographic dependence between LHI and MR (and possibly LHI and ER), yet high levels of inferred self-replenishment at all four locations; (iv) variable genetic diversities: low mtDNA genetic diversity at all three locations within the western region, but not at the peripheral location; and (v) high msatDNA genetic diversity at all four locations.Understanding both time and spatial scales of gene flow and the levels of genetic diversity is vital to determine best practice management, maximize biodiversity conservation, and evaluate the capacity of coral reef fishes to recover should they become locally extinct. In this study, C. tricinctus with the exception of two clades, one of which consisted exclusively of the peripheral location. The lack of geographic population structure within the western region may result if a small number of recruits per generation maintain spatial genetic homogeneity  indicate that the three locations within the western region and the peripheral location are genetically differentiated. Although high mtDNA gene flow may provide some assistance to distant populations through recolonization following local extinctions and increasing genetic diversity . The strong discrepancy between mtDNA and contemporary levels of gene flow in C. tricinctus is increasingly being documented in other coral reef fishes such as snappers Lutjanus carponotatus , a slow recovery time is expected following population declines, due to intermittent pulse replenishment.The estimation of contemporary gene flow is important for conservation because models predict that a few recruits per generation over evolutionary time scales will not sustain populations  are highly similar to the estimated levels of self-recruitment in other congeneric butterflyfish in Papua New Guinea , ER being the exception. Similar genetic diversities have been found in other coral reef fish using cytochrome b including the endemic Hawaiian butterflyfishes Chaetodon fremblii, C. miliaris, and C. multicinctus  but extremely low abundance of C. tricinctus individuals, it is likely that self-recruitment is limiting population numbers. If unique genetic diversity is a feature of NI populations of endemics within the region, then protecting these populations and the habitats they rely on is vitally important.C. tricinctus, the MPAs at the western region are of limited benefit to the unprotected peripheral location (NI). Therefore, the peripheral location requires some protective management strategies to conserve its genetically unique population of C. tricinctus. However, within the western region, LHI is an important source of gene flow to both MR and ER and as such, warrants continued MPA protection and monitoring. Similar patterns of gene flow between locations has also been found for the endemic Mcculloch's anemonefish, A. mccullochi  in


Quercus robur) half-sib families. We quantified genetic diversity at the plot level by genotyping all oak saplings and assessed overall damage caused by ectophagous and endophagous herbivores along a gradient of increasing genetic diversity. Damage due to ectophagous herbivores increased with the genetic diversity in oak sapling populations as a result of higher levels of damage in mixtures than in monocultures for all families (complementarity effect) rather than because of the presence of more susceptible oak genotypes in mixtures (selection effect). Assemblages of different oak genotypes would benefit polyphagous herbivores via improved host patch location, spill over among neighbouring saplings and diet mixing. By contrast, genetic diversity was a poor predictor of the abundance of endophagous herbivores, which increased with individual sapling apparency. Plant genetic diversity may not provide sufficient functional contrast to prevent tree sapling colonization by specialist herbivores while enhancing the foraging of generalist herbivores. Long term studies are nevertheless required to test whether the effect of genetic diversity on herbivory change with the ontogeny of trees and local adaptation of specialist herbivores.A growing body of evidence from community genetics studies suggests that ecosystem functions supported by plant species richness can also be provided by genetic diversity within plant species. This is not yet true for the diversity-resistance relationship as it is still unclear whether damage by insect herbivores responds to genetic diversity in host plant populations. We developed a manipulative field experiment based on a synthetic community approach, with 15 mixtures of one to four oak ( Over the last decades, the role that biodiversity plays in ecosystem functioning has emerged as a key issue in ecology i.e. associational resistance) when the presence of non conspecific neighbours (i) reduces host plants concentration and the probability to be located by specialist herbivores vs biomass removed) The diversity – resistance hypothesis states that species rich plant communities suffer less feeding damage by herbivores than plant monocultures i.e. genetic diversity) is a key component of biodiversity. Recent research in the field of community genetics has shown that host plant genotype is one of the ecological filters shaping the structure of insect species assemblages et al.Intraspecific diversity  and (ii) the magnitude of the effect depends on host specialization of insect herbivores, being higher for more generalist species. To test these hypotheses we designed a common garden experiment with 90 synthetic mixtures of oak saplings composed of one to four half-sib families. We genotyped all saplings and evaluated the amount of damage caused by ectophagous insect herbivores  and endophagous leaf miners  on each individual sapling. We assessed the level of genetic diversity in each mixture and estimated the correlation between diversity and insect herbivory.With a few exceptions No specific permits were required for the described field studies. The site on which the experimental common garden was established is owned by our institute (INRA) and is no subjected to any protection scheme. This work did not involve any endangered or protected species or area.Quercus robur), referred to hereafter as ‘mother trees’, sampled at random within a 10 km radius at a site 40 km south of Bordeaux . In March 2008, we sowed the acorns at the nursery of the forest research centre of the French National Institute for Agricultural Research (INRA), to produce four half-sib families of oak seedlings. The seedlings were grown in individual pots containing peat and were treated with fungicide and insecticide during the first growing season (i.e. 2008), to prevent damage before planting. In March 2009, the seedlings were transplanted to a clearing surrounded by pine trees (Pinus pinaster) and broadleaved species .In autumn 2007, we collected acorns from the canopy of four mature pedunculate oaks , 2 (n = 6), 3 (n = 4) and 4 (n = 1) families per plot. Each plot contained four rows of three seedlings; the seedlings were 0.2 m apart and the plot area was 0.24 m2 (0.60×0.40). Within each plot, oak families were planted at equal density in a regular alternate pattern, such that seedlings from the same family were never adjacent in mixed plots. The plots were separated by a distance of 3 m and were randomly distributed within the blocks. Blocks were 14 m ×6 m in size and were located 4 m apart .The experimental site was fenced to prevent grazing by mammalian herbivores. The herbaceous plants growing between plots were removed by mowing, twice yearly. Pine bark chips were spread on the soil of each plot to control the vegetation and limit evaporation. Plots were watered during the summer of 2009, to minimise seedling mortality. In August 2011, 25 out of the 1080 planted seedlings were dead , skeletonisers (adult grasshoppers and Tenthredinoidea larvae), rollers (mostly Lepidoptera larvae) and miners (mostly Microlepidoptera larvae). No gall makers were observed. The percentage leaf area affected was visually estimated for each leaf and each guild using six classes  and then averaged per sapling.Herbivore damage on oak leaves was assigned to four different trophic guilds: et al.Q. robur are polyphagous species able to consume plant tissues from different genera and families and may be then considered as generalists  developed by Guichoux et al.All oak saplings and the four mother trees were genotyped with 12 microsatellite markers . More detailed information about genetic structure of the oak seedlings population is provided in We initially used the number of maternal lineages per plot as a measure of genetic diversity. However, as a given mother tree could have been pollinated by several father trees, the offspring may be half-sibs or full-sibs and the proportion of the two types of saplings could vary within families and within sapling assemblages. The number of maternal lineages per plot may therefore underestimate genetic diversity and be poorly correlated with variation in insect damage.genetic diversity (GD) refers to the number of maternal lineages per plot, whereas genetic relatedness (GR) refers to the mean between-saplings relatedness per plot.We then determined SSR genotypes, to calculate the genetic relatedness between oak saplings, thereby improving estimates of genetic diversity per plot and switching from an almost categorical  to a more continuous  variable. Hereafter, i.e. 0.5 for full sibs, 0.25 for half sibs and 0 for unrelated saplings). GR was calculated for all pairs of individuals (n = ½(1016×(1016–1))  = 515,620 pairs) and we used these values to calculate a mean genetic relatedness for each plot. Mean genetic relatedness significantly differed between plots with different numbers of maternal lineages , decreasing with increasing number of lineages , mean relatedness was averaged across a variable number of individual saplings per plot (9 to 12). For the sake of consistency, missing genotypes were also removed before the analysis of insect damage data. The final dataset contains 1002 individuals (6 blocks ×15 plots ×12 trees –25 dead saplings –53 unamplified or mismatched genotypes).i.e. herbivory by ectophagous insects, abundance of leaf miners and sapling height) were analysed using each individual tree as a replicate to make it possible to test for the possible effects of interactions between mother tree identity (MT) and GD or GR. We accounted for spatial replication by nesting the ‘population effect’ (i.e. 1 population  = 1 plot  = 12 saplings) within the block effect, both factors being treated as random effects in all mixed models, in order to specify that individual observations were correlated within blocks and within plots.Response variables .i.e. subtracting the sample mean from all observations) and reduced (i.e. dividing centred variables by their sample standard deviation) in order to make model coefficients comparable within and between models In order to determine potential genetic effects on sapling height, we first performed two sets of linear mixed models with MT and GD or GR, separately, and their interactions as fixed effects. We then carried out linear mixed models to test the effect of sapling height, MT, GD or GR, and their interactions on herbivory by ectophagous insects (% leaf area damaged) and by endophagous insects (abundance of leaf miners), separately. Prior to analyses, continuous explanatory variables  were centred  for generalised linear mixed models performed on the abundance of leaf miners. Log-likelihood R2 values were calculated to estimate the amount of variance explained by each independent variable Test statistics for fixed effects were based on F values for linear mixed models (herbivory by ectophagous insects and sapling height) and on χlme procedure logit function lmer procedure in the lme4 package in R Data for sapling height and damage due to ectophagous insects were analysed with linear mixed models with the et al.i (RFiO) as the ratio of the damage observed on each family i (i from 1 to 4) in a mixture (iC) to that observed on this family in monoculture (iM) We used the method developed by Loreau and Hector i (RFiE) under the null hypothesis (i.e. no effect of genetic diversity on damage) was simply its proportion in the mixture, i.e. 1/n where n is the number of families in the mixture The expected relative forage of the family The deviation of the observed relative damage in a mixture from the relative damage expected in the corresponding monoculture was thus:The total observed damage in the mixture was calculated as:The total expected damage in the mixture was calculated as:A positive NGDE indicates associational susceptibility (higher level of damage observed in mixtures than expected from mean damage levels in the corresponding monocultures), whereas a negative NGDE indicates associational resistance (lower level of damage observed in mixtures than expected from mean damage levels in the corresponding monocultures).The NGDE can be further divided into two additive components: a complementarity effect (CE) and a selection effect (SE) iΔRC over all families at the plot level:The CE is assessed by calculating the mean i.e. when oak families are, on average, consumed more in mixtures than it would be expected from their consumption in monocultures.CE measures the change in mean relative forage of the species. CE is positive when the mean relative forage increases i and Mi:The calculation of SE takes into account the covariance between ΔRCSE values are used to determine whether there is a relationship between consumption in the monoculture and relative forage in mixtures. SE is positive when plant species that are consumed in larger amounts in monocultures (less resistant) also have higher relative forage values in mixtures, thus making a greater contribution to total plot damage.NGDE, CE and SE were calculated for all levels and combinations of mixtures within each block, giving a total of 66 comparisons between observed and expected values. The significance of each effect  was determined by one sided t-tests F3,909  = 2.88, p = 0.035) but we observed no significant effect of genetic diversity  or genetic relatedness  on sapling height.Mean sapling height significantly differed between oak families , GD×H  and GR×H  interactions had no significant effect on damage , but not by interactions between these factors . Signifin damage .i.e. leaf miners), sapling height emerged as the main factor determining their abundance on individual saplings . The coefficient estimate of GD×H interaction term was negative . Both complementarity and selection effects (CE and SE) were significantly different from zero , herbivory in mixtures was much higher than expected (The net genetic diversity effect (NGDE) on herbivory by ectophagous insects was overall significantly positive , indicatrom zero  but had rom zero : mean CErom zero , Table 2rom zero . For famexpected .There was no significant NGDE or CE on the abundance of endophagous herbivores . By contNGDE and CE were not significant for sapling height, for either the grand mean, or for any of the levels of genetic diversity, with exception of the 4-families mixtures . SE was Based on a large number of samples and a manipulative experiment this study shows for the first time that genetic diversity can trigger associational susceptibility to insect herbivory vs. other ecological factors as drivers of ecosystem processes is a central issue for community genetics et al.The relative importance of genetic diversity et al.Quercus robur, but they did not measure corresponding herbivory. We are aware of only two studies that investigated the relationship between tree genetic diversity and insect damage. They reported a trend towards higher levels of pest damage in monocultures than in mixtures of willow clones Quercus and the Castanea genera while all the ectophagous insects we observed in the field were polyphagous species able to feed on host plants belonging to different families  has been initially attributed to a spill over of generalist herbivores from their preferred host plants to nearby suitable but less suitable host plants et al.The higher damage by herbivorous insects in plant species mixtures  and medium-sized (median of heights distribution) saplings tended to increase with genetic diversity at the plot level  had a significant effect on the abundance of specialist herbivores (leaf-miners). This finding is consistent with previous studies showing that genotype Phyllonorycter sp. and Stigmella sp.). Separate analyses of each species of leaf miner would have generated too many zero counts, so we decided to pool data into a single category of “insect specialists”. However, Tack and Roslin Phyllonorycter sp. and Stigmella sp. responded differently to genetic and environmental treatments. Considering the abundance of several leaf-miner species together may therefore have prevented the detection of species-specific abundance patterns. Further investigation, after the oak saplings have been colonised by a larger number of insect species, are required for more detailed comparisons of the responses of generalist and specialist herbivores to genetic diversity.The mean abundance and species richness of leaf miners were very low in our experiment, with abundance scores of 0 to 10 mines in 20 leaves per sapling and 85% of total abundance represented by only two of the nine observed species (i.e. positive complementarity effect) and (ii) an increased damage exposure of individuals from more resistant genotypes in the vicinity of individuals from more susceptible genotypes (i.e. negative selection effect). To date many studies have shown that herbivore diversity increases with host plant genetic diversity Unlike plant species richness, plant genetic diversity may not provide sufficient functional contrast to prevent host colonization by specialist herbivores while enhancing the foraging of generalist herbivores. Overall, we observed a significant effect of tree genetic diversity on generalist herbivores but not on specialists. Increasing genetic diversity resulted in higher damage by generalist herbivores because of (i) a general increase in leaf consumption in more diverse genotype mixtures Click here for additional data file.Figure S2Effect of the number of half-sib families per plot  on the mean genetic relatedness among oak seedlings within plots . Open circles represent individual plots (n = 90); filed circles represent mean genetic relatedness per level of genetic diversity.(DOCX)Click here for additional data file.Figure S3Negative selection effect of genetic diversity on exophagous herbivores.(DOCX)Click here for additional data file.Figure S4Effects of genetic diversity on oak height heterogeneity within plots. Each dot represents the mean difference (± SE) between the 75th percentile and the median (50th percentile) of sapling heights distribution within plots. This difference indicates how far taller trees were apparent to herbivores within plots.(DOCX)Click here for additional data file.Table S1Summary of genetic data describing the genetic structure of the population used to constructed experimental plots.(DOCX)Click here for additional data file.