The AI seroprevalence was significantly higher in geese (21.0%) than in ducks (11.7%) (P<0.001). Besides, the spatial distribution of AIVs was not homogeneous as significant differences among regions were observed. The prevalence of antibodies against AIVs subtypes H5 and H7 were 1.1% and 0.3%, respectively, using hemagglutination inhibition test (HI). The overall and H5 seroprevalences slightly increased after the hunting period , while the H7 seroprevalence remained at the same level (0.3%). The proportion of flocks infected by AIVs was 65.3%, while 11.2% and 4.9% of flocks were positive for H5 and H7, respectively. Viral shedding was not detected in any of the 47 samples positive by both ELISA and HI, tested by RRT-PCR. The individual incidence after the hunting season was 3.4%. The fact that 57 animals seroconverted, 15 of which were confirmed by HI (12 H5 and 3 H7), was indication of contact with AIVs during the hunting period. The results indicate that waterfowl used as decoys are frequently exposed to AIVs and may be potentially useful as sentinels for AIVs monitoring. The seroprevalence detected and the seropositivity against AIVs H5 and H7, suggest that decoys can act as reservoirs of AIVs, which may be of animal and public health concern.A longitudinal study was carried out to determine the seroprevalence of avian influenza viruses (AIVs) in waterfowl used as decoys in Andalusia, southern Spain. A total of 2319 aquatic birds from 193 flocks were analyzed before and after the hunting season 2011–2012. In the first sampling, 403 out of 2319 (18.0%, CI Avian influenza viruses (AIVs) are among the most important emerging zoonotic pathogens worldwide, affecting a wide variety of avian and mammalian species, including humans Podiceps cristatus) found dead in the Basque Country (Northern of Spain) Due to the strategic location on the migratory flyway of wild birds between Eurasia and Africa, the high number of wetlands and the diversity of wild bird species, Spain is considered a risk area for HPAIVs introduction Small game is considered an important economic activity in Spain. Waterfowl are used as decoys and present great relevance in certain regions with presence of wetlands. Decoys are domestic waterfowl species, including Anseriformes and Charadriiformes, reared by hunters in their backyards, which act as lures for hunting purposes . The use of decoys in hunting results in frequent contact with wild birds, and therefore a high risk of diseases transmission, not only from wild bird to decoys but also from decoys to human The aim of the present study was to determine the prevalence and incidence of AIVs in aquatic birds used as decoys in Andalusia, before and after the hunting period 2010–2011, and to detect circulation of H5 and H7 subtypes in their populations.This study complies with the current laws regarding ethics and animal use for scientific purposes in Andalusia where samples were gathered. All necessary licenses were obtained for this study. Permits for the captures and blood collections of decoys were provided by the Regional Government of Andalusia, Spain (Register number: BOJA-246/2010). No ethical approval was deemed necessary as this study did not involve killing of animals. Data and samples were obtained by official veterinarians from Consejería de Agricultura, Pesca y Desarrollo Rural of the Regional Government of Andalusia, and handling procedures and sampling frequency were designed to reduce stress and health risks for subjects, according to European (86/609) and Spanish laws .g for 15 min. Then, serum was separated and stored at −20°C until analyzed. Moreover, data on sampling date, flock location, census, individual identification and species (ducks or geese) were recorded.The longitudinal study was conducted in Andalusia, located in southern Spain  . AndalusThe presence of antibodies against the nucleoprotein of AIVs type A was determined using a commercial blocked enzyme linked assay (ELISA)  according to the manufacturer’s recommendations. Sera positive against AIVs type A were then analyzed by means hemagglutination inhibition test (HI) as previously described After serological results, a convenience sampling was carried out to detect AIVs shedding from birds positive by ELISA. A total of 47 cloacal swabs (34 from the first sampling and 13 from the second), from 44 selected seropositive birds by ELISA were collected. Nine of the 44 seropositive birds were also positive against H5 by HI test. Samples were analyzed using an one-step real time reverse transcription polymerase chain reaction (RRT-PCR) with Taqman specific for the matrix gen (gene M) in the segment 7 of AIVs using primers previously described http://CRAN.R-project.org/package=lme4). Inference was based on model comparison of nested models (ANOVA), and the process of model selection was based on the lowest Akaike information criterion (AIC) value. Statistical analyzes were carried out in R software (http://www.r-project.org/).The prevalence of antibodies against AIVs was estimated from the ratio of positive to the total number of samples, with the exact binomial confidence intervals of 95% 95%: 15.9–19.0) birds tested by ELISA during the first sampling  and 0.3% , respectively . The individual seroprevalence against H5 and H7 subtypes were 1.1%  had at least one positive animal by ELISA . The serThe individual seroprevalence increased slightly after the hunting season . A total of 209 animals (three seropositive and 206 seronegative decoys in the first sampling) could not be sampled in the second sampling. Results for HI were similar to those obtained in the first sampling, with 28 birds serologically positive to H5  and seven to H7 . However, 66 decoys positive in the first sampling showed negative results in the second sampling, while 57 individuals seroconverted after the hunting period ; 12 of them were confirmed as positive against H5 and three against H7 by HI.The flock prevalence after the hunting period  was very similar to that found in the first sampling . HoweverAIVs RNA was not detected in any of the 47 cloacal swabs analyzed by RRT-PCR, including three animals positive against H5.Even though wild and domestic birds have been the main target of surveillance programs and AI investigations, studies on the prevalence of AIVs birds reared in backyard are very limited in Europe Active circulation of AIVs has been previously detected in wild and domestic waterfowl in Spain  [15], [1Anser albifrons) and Emperor geese (Anser canagica) had the highest prevalences, respectively Even though most of the decoy flocks analyzed (70.6%) were mixed, including geese and ducks, and individuals were bred with similar management conditions, a significantly higher seroprevalence was found in geese compared to ducks. Differences in the prevelence of AIVs among species have been previously described and could be associated to variations in the immunological response, behavior, density-related patterns or gregariousness patterns. In the majority of studies The flock seroprevalence levels indicate widespread circulation of AIVs in decoys in Andalusia. However, the results show that the spatial distribution of AIVs in decoys in Andalusia was not homogeneous. Statistically significant differences in seroprevalence were observed among municipalities, with highest seroprevalences in the regions located close to the river Guadalquivir (Cádiz and Seville), the largest river in Andalusia. The seropositivity obtained in Huelva (6.1%) was similar to that previously detected by Astorga et al. (1994) in Doñana National Park, the main wetland in Spain, and within the route of different migratory species between Europe and Africa. Environmental factors have been associated with the risk AIVs transmission The individual incidence between the first and the second sampling was 3.4%. A total of 57 individuals seroconverted, which confirms AIVs infections during the study period. The results may be associated to contact with infected decoys, migratory and resident infected wild birds or to the presence of AIVs in the environment where decoys are kept during the hunting activity The detection of antibodies against H5 and H7 by HI indicates that birds were exposed and responded serologically to the contact with these AIVs subtypes. In fact, 15 out of the 57 seroconverted decoys analyzed in the second sampling were confirmed by HI as positive against H5 (12) and H7 (3), indicating recent infections with both subtypes. Although the pathogenicity of the circulating strains could not be determined, the results indicate that decoys could represent a risk for HPAIVs emergence associated to genetic mutations, as has been previously demonstrated In conclusion, the results of the present study confirm a widespread circulation of AIVs in waterfowl species used as decoys in Andalusia. The seroprevalence obtained indicates that decoys are frequently exposed to AIVs and may potentially be useful as sentinels for AIVs monitoring. The seropositivity against AIVs H5 and H7, suggest that decoys can act as reservoirs of these subtypes, which may be of animal and public health concern. Because of the direct contact among decoys, wildlife and human, these species should be considered as risk species for the transmission of bird-borne pathogens, including AIVs. Control measures to limit transmission from wild birds to decoys as well as from decoys to wild birds should be implemented.


Hybridization between domestic and wild animals is a major concern for biodiversity conservation, and as habitats become increasingly fragmented, conserving biodiversity at all levels, including genetic, becomes increasingly important. Except for tropical forests and true deserts, African wildcats occur across the African continent; however, almost no work has been carried out to assess its genetic status and extent of hybridization with domestic cats. For example, in South Africa it has been argued that the long-term viability of maintaining pure wildcat populations lies in large protected areas only, isolated from human populations. Two of the largest protected areas in Africa, the Kgalagadi Transfrontier and Kruger National Parks, as well as the size of South Africa and range of landscape uses, provide a model situation to assess how habitat fragmentation and heterogeneity influences the genetic purity of African wildcats. Using population genetic and home range data, we examined the genetic purity of African wildcats and their suspected hybrids across South Africa, including areas within and outside of protected areas. Overall, we found African wildcat populations to be genetically relatively pure, but instances of hybridization and a significant relationship between the genetic distinctiveness (purity) of wildcats and human population pressure were evident. The genetically purest African wildcats were found in the Kgalagadi Transfrontier Park, while samples from around Kruger National Park showed cause for concern, especially combined with the substantial human population density along the park's boundary. While African wildcat populations in South Africa generally appear to be genetically pure, with low levels of hybridization, our genetic data do suggest that protected areas may play an important role in maintaining genetic purity by reducing the likelihood of contact with domestic cats. We suggest that approaches such as corridors between protected areas are unlikely to remain effective for wildcat conservation, as the proximity to human settlements around these areas is projected to increase the wild/domestic animal interface. Thus, large, isolated protected areas will become increasingly important for wildcat conservation and efforts need to be made to prevent introduction of domestic cats into these areas. Despite international conservation interventions, global biodiversity continues to decline  and their wild ancestors suggests little or no reproductive barriers may exist (Driscoll F. s. silvestris) confirm high levels of admixture with domestic cats in Hungary and Scotland  were obtained as dried tissue material from various museums throughout South Africa . Due to the importance of Kgalagadi Transfrontier Park (KTP) as a large protected area supporting a high African wildcat population, 47 samples were collected within the KTP, and 10 samples outside KTP, either as road kill or by trapping, between April 2003 and December 2006 ; first, historical collections  diluted to a final concentration of 20 ng/μL and stored at −80°C.DNA from tissue material was extracted using the Cell Lysis stock solution  and phenol–chloroform–isoamylalcohol (Sigma-Aldrich). DNA from hair samples was extracted with 200 mmol/L NaOH and 200 mmol/L HCl, 100 mmol/L Tris–HCl, pH 8.5 Herbst . DNA from MgCl2, primers at specific concentrations (Table S2), with the final reaction volume adjusted to 10 μL with distilled water. All multiplex reactions were amplified using the following thermal cycle: an initial denaturation at 95°C for 3 min, followed by 30 cycles of initial denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s. A final extension was carried out at 72°C for 15 min. Successful amplification was verified using agarose gel electrophoresis. Purified PCR fragments were separated on an ABI Prism 3100 Genetic Analyzer , using GENESCAN™-500 (-250) as an internal size standard (Appl-ied Biosystems). Allele sizes were visualized and scored using GENEMARKER v1.95 .We selected 13 unlinked microsatellite markers previously characterized in domestic cats  and ran five independent models for each value of K. Each model consisted of 1,000,000 generations of which the first 100,000 were discarded as burn-in. We also applied the admixture model with correlated allele frequencies as we suspected hybrid individuals to be present in the dataset based on morphological observations. The optimal K value was determined using the method described by Evanno et al.  was estimated using Bayesian assignment tests implemented in STRUCTURE v2.3.4  of each individual's multilocus genotype (I) derived from each of the predefined K number of clusters. Therefore, while individual genotypes may show membership to more than one cluster , the sum of its qik is always one. These assignment values can therefore be useful in the identification of hybrid individuals. However, the proportion of pure and admixed (hybrid) individuals within a given sample will be strongly influenced by the validity of the assumed priors and the efficiency of analyzed loci used in the Bayesian analysis, and cannot be statistically tested . 95% CI intervals for qik – values obtained from simulated genotypes were determined in R (R Core Development Team 2010) for each scenario to assess the efficiency of the admixture analysis to detect the different classes of F1, F2, and backcrossed genotypes in our data.STRUCTURE calculates assignment values as the proportion . Using these assignment values and their standard deviations, we were able to class individual genotypes as genetically pure (African wildcat or domestic cat), F1, F2, or F1 backcrossed (Table S1). We also calculated the distance of all sample sites from the boundaries of formally protected areas as described by SANBI . Sites b(F. silvestris) are limited to short-term studies, small sample sizes, and opportunistic observations  were radio collared from 2003 to 2006 (46 months) (see Table S3 for more details). Home ranges were calculated using minimum convex polygons (MCP) Mohr , and oven = 3] and males = 657 h [n = 5]).Two types of data were collected: (1) radiolocation observations, when only a radio-fix of the animal was recorded, and (2) continuous observations, when radio collared African wildcats were followed by a vehicle for varying periods of 1–14 h . A rotation system was followed in order to obtain equal observation records for all cats. Over the course of the study 1538 h were spent with habituated wildcats  to human infrastructure and settlement density was assessed using the Global Human Footprint (GHF) Dataset of the Last of the Wild Project . Cats from the Kgalagadi Transfrontier Park assigned highly to the African wildcat genetic cluster (mean qAWC = 0.982). Surprisingly, based on morphology, seven suspected hybrids assigned very highly to the African wildcat genetic cluster (qAWC ≥ 0.89) and four to the domestic cat genetic cluster (qDC ≥ 0.85) . Of the putative African wildcats collected outside protected areas (n = 68), seven individuals had admixed genotypes (qDC: 0.23–0.79) while two cats assigned highly to the domestic cat cluster (qDC ≥ 0.94). The STRUCTURE results were supported by scatter plots in the PCA that separated genetic groups  represent pure domestic (n = 5) and wildcats (n = 1), while other putative wildcats (n = 7) are hybrid and possibly backcrossed individuals  , compared to wildcats from outside protected areas  km2 in adult female African wildcats. The annual home range sizes (95% MCP) of adult male cats were significantly larger (between 1.6 and 2.2 times) than female wildcats . Female cats displayed extensive overlap of home ranges (average of 33.4 ± 13.4%), while the overlap between male home ranges was limited (average of 3.5 ± 5.3%), with no overlap of core areas. Males overlapped extensively with the home ranges of up to four females.Annual home range estimates (MCP 95%) for adult males was 7.7 (±3.5) kmP = 0.0003), standard deviation within zones (P = 0.0097), and distance to the nearest town  and genetic diversity and structure, rather than admixture analysis.Our genetic analyses of African wildcat collections spanning five decades, and from regions throughout South Africa, indicate high genetic distinctiveness from their domestic counterparts, with seemingly limited hybridization and introgression. This finding is maybe surprising given the genetic status of wildcats elsewhere in the world  indicating possible F2 offspring or even wildcat backcrosses, which may obscure morphological features distinguishing hybrids from parental phenotypes. This may also present problems where well-meaning members of public keep “wildcats” for breeding and reintroduction into the wild, or as pets in areas where wildcats are present, by increasing the chances for contact and breeding with wild populations.Despite the genetic distinctiveness of African wildcat populations throughout South Africa, we identified a few genetic anomalies to this overall pattern. First, we identified seven putative wildcat specimens that showed varying levels of admixture, most likely resembling F1 or F2 hybrids and possibly backcrosses  is still genetically pure, with no signs of hybridization and introgression (but see Herbst Even when hybridization is prevalent and widespread within a species, it might be locally rare (Oliveira et al.


Synaesthesia is a condition in which one property of a stimulus triggers a secondary experience not typically associated with the first . Recent work has explored a variety of cognitive and perceptual traits associated with synaesthesia. One example is in the domain of personality, where higher rates of positive schizotypy and openness to experience and lower agreeableness have been reported in synaesthetes who experience color as their evoked sensation relative to typical adult controls. Additionally, grapheme-color synaesthetes have previously been reported to show elevated mental imagery compared to typical adults. Here, we aimed to further elucidate the relationship between personality, synaesthesia, and other cognitive traits. In Study 1, we examined self-reported schizotypy and self-reported visual imagery vividness in grapheme-color synaesthetes and typical adults. Our results partially replicated previous findings by showing that synaesthesia was associated with greater positive schizotypy and enhanced self-reported imagery vividness. The results also extend previous reports by demonstrating that differences in positive schizotypy and mental imagery vividness are not related in grapheme-color synaesthesia. In Study 2, we sought to build on prior work showing lower agreeableness and increased openness to experience in synaesthetes by examining whether grapheme-color synaesthesia is associated with other conceptually related traits; namely lower self-monitoring and increased sensation seeking. We did not find any differences between synaesthetes and controls on either of these traits. These findings are discussed in relation to potential factors that may contribute to the observed personality profile in grapheme-color synaesthesia. Synaesthesia is a condition in which stimulation in one modality leads to involuntary secondary sensations within the same or different modality . For exaThere is also some evidence suggesting that synaesthesia might be linked to a specific personality profile. For example, color synaesthesia has been recently associated with positive and disorganized schizotypy . SchizotSynaesthetes who experience color as their concurrent  have also been found to score higher on the openness to experience, and lower on agreeableness subscales of the Big Five Inventory . These sIn the general population, a number of other traits have been associated with personality dimensions that have been shown to be different in color synaesthetes . For example, sensation seeking and openness to experience both relate to the desire to seek novel and exciting sensations and experiences. With this in mind, the primary objectives of the current study were as follows. Firstly, we aimed to replicate previous findings linking synaesthesia for color with enhanced self-reported positive and disorganized schizotypy , and menM = 38.6 SD = 15.05) and 35 age and gender matched controls  took part in this study. Some of the synaesthetes also reported spatial forms (7), personification (6), mirror-touch (3), and other forms of synaesthesia involving color (16). They were recruited from a database of verified synaesthetes. We invited 110 synaesthetes to take part, 11 of whom had previously taken part in the study by Thirty-five grapheme-color synaesthetes  = 3.697, p = <0.001, Cohen’s d = 0.88] , indicating higher levels of positive schizotypy in synaesthetes; therefore replicating one aspect of previous findings (t(68) = 2.160, p = 0.034, Cohen’s d = 0.51] replicating previous findings .Only the UEs subscale of O-Life and Total VVIQ (TVVIQ) scale were normally distributed. Both UE and TVVIQ had high consistency scores with Cronbach’s Alpha of 0.884 and 0.972, respectively. Both UE and TVVIQ were then analyzed using separate independent samples findings . Synaestfindings   = 389.500, z = -2.622, p = 0.009, r = -0.31] where synaesthetes reported greater imagery than controls. There were no other statistically significant differences (Introvertive Anhedonia: U(70) = 651.500, z = 0.460, p = 0.646, r = 0.05; Cognitive Dizorganization: U(70) = 692.000, z = 0.935, p = 0.350, r = 0.01; Compulsive Non-Conformity: U(70) = 674.00, z = 0.726, p = 0.468, r = 0.08; VVIQ closed eyes: U(70) = 497.50, z = -1.352, p = 0.177, r = -0.16).All other subscales, which were not normally distributed, were analyzed with non-parametric independent-samples Mann–Whitney r(35) = -0.169, p = 0.330] or controls .To examine the relationship between elevated schizotypy and visual imagery scores in synaesthetes, we also calculated Pearson Correlation Coefficients. The correlation between TVVIQ and UE was not significant in either synaesthetes  and negatively associated with imagery  = -2.15, p = 0.034]. The results also showed that the association between imagery and schizotypy was not significant  = -0.98, p = 0.330]. The mediation analysis did not show a mediating role of imagery on the relationship between group and schizotypy ; model statistics between group and schizotypy when controlling for imagery were B = 5.02, t(67) = 3.32, p = 0.001. In this regard, elevated self-reported visual imagery did not mediate the relationship between increased positive schizotypy and presence of synaesthesia for color.Additionally, we ran a mediation analysis using bootstrapping with bias-corrected confidence estimates  to assesFigure 4). Again, there was a negative association between group and TVVIQ , and a positive association between group and schizotypy . There was no significant association between schizotypy and imagery . Although the significance level of the relationship between group and imagery was reduced when controlling for schizotypy , the mediation effect was not significant suggesting that schizotypy is not a significant mediator of the relationship between group and imagery .An additional mediation analysis was run in order to determine whether the presence of schizotypy would mediate levels of imagery using the same method as above  were invited to take part in this study. From this 37 grapheme-color synaesthetes  and 37 age and gender matched controls  took part. Some of the synaesthetes also reported spatial forms (4), personification (3), and mirror-touch (2) synaesthesia, and other forms of synaesthesia involving color (9). For all synaesthetic participants, synaesthesia had been confirmed either using an online consistency test  = -1.268, p = 0.209, Cohen’s d = -0.29]. Similarly, no significant differences were found between the groups on the Sensation Seeking subscales using Independent-Samples Mann–Whitney U tests  (see Table 1 for more statistics).Only the SM scale was normally distributed. Cronbach’s alpha for this measure was 0.771. Independent samples This study examined the relationship between increased self-reported mental imagery and schizotypy in grapheme-color synaesthesia. Building on prior work indicating that greater positive and disorganized schizotypal traits amongst individuals with synaesthesia for color  and incrOur findings partially replicate One possible reason for elevated positive schizotypy in synaesthesia for color may be that there is some degree of similarity in mechanisms contributing to synaesthetic, and the unusual visual experiences associated with positive schizotypy. For example, synaesthesia and schizophrenia have both been linked to abnormalities in early stages of visual processing e.g., . In schiAdditionally, as this study tested grapheme-color synaesthetes  on vividness of visual imagery we can only make inferences about visual forms of synaesthesia and imagery within the visual domain. Another question is whether the number of synaesthesia forms could influence current results. Based on prior findings suggesting an association between the number of synaesthetic modalities and vividness of reported imagery  it couldBased on previous findings demonstrating an association between synaesthesia and greater openness to experience , and stuIn light of previous research showing an association between synaesthesia and decreased agreeableness , it was In sum, we demonstrated that grapheme-color synaesthetes show greater positive schizotypy and self-reported visual imagery vividness compared to controls. By examining these two constructs in one sample of synaesthetes, we also found that heightened positive schizotypy is not a result of a relationship between schizotypy and mental imagery vividness. Finally, synaesthetes did not differ from controls in terms of levels of SM or sensation seeking despite previous work reporting that color synaesthetes differ from controls on agreeableness and openness to experience , suggestAJM collected and analayzed the data. The authors contributed equally in all other aspects.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Melursus ursinus) are endemic to the Indian subcontinent. As a result of continued habitat loss and degradation over the past century, sloth bear populations have been in steady decline and now exist only in isolated or fragmented habitat across the entire range. We investigated the genetic connectivity of the sloth bear meta-population in five tiger reserves in the Satpura-Maikal landscape of central India. We used noninvasively collected fecal and hair samples to obtain genotypic information using a panel of seven polymorphic loci. Out of 194 field collected samples, we identified 55 individuals in this meta-population. We found that this meta-population has moderate genetic variation, and is subdivided into two genetic clusters. Further, we identified five first-generation migrants and signatures of contemporary gene flow. We found evidence of sloth bears in the corridor between the Kanha and Pench Tiger Reserves, and our results suggest that habitat connectivity and corridors play an important role in maintaining gene flow in this meta-population. These corridors face several anthropogenic and infrastructure development threats that have the potential to sever ongoing gene flow, if policies to protect them are not put into action immediately.Sloth bears ( The importance of maintaining meta-populations of large carnivores in a landscape with habitat connectivity between source populations is an intuitive and logical concept supported by a large body of scientific evidence –3. SmallMelursus ursinus) is a large wide-ranging carnivore species endemic to the Indian subcontinent . We used a previously optimized panel of seven microsatellite loci  that hadIn order to improve amplification success, we used a two-step PCR amplification procedure . In the 2, 1X Gold buffer, 0.1 mM of each dNTP, 2 μl 10X BSA, and 4–5 μl of the DNA extract. PCR conditions were: initial denaturation step (95°C for 10 min), 25 cycles of denaturation (95°C for 30 sec), annealing for 30 sec (60°C for brown bear primers and 50°C for the remaining primers in the pre-amplification step), and extension (72°C for 1 min), followed by a final extension step (72°C for 7 min). The second PCR: this locus-specific PCR consisted of 10 μL reactions which contained 0.5 U Ampli Taq Gold DNA Polymerase, 2 mM MgCl2, 1X Gold buffer, 0.1 mM of each dNTP, 2 μL 10X BSA, 0.2 μM of both forward and reverse primer and 1.5 μl of pre-amplified product as the DNA template. The PCR conditions for this locus-specific reaction were same as the pre-amplification, except that the annealing temperature was specific for each primer, and we amplified the product in 45 cycles instead of 25 cycles. We ran all PCR products on an ABI 3730xl sequencer, using GeneScan–500 LIZ (Applied Biosystems) size standard. We scored alleles using GeneMapper-4.1 (Applied Biosystems).Pre-amplification PCR: this PCR consisted of 25 μl reactions containing 0.5 U Ampli Taq Gold DNA Polymerase , 0.01 μM each fluorescently-labeled forward primer, 0.01 μM each unlabeled reverse primer, 2 mM MgClIn order to control DNA quality, we used a two-step approach to select good quality samples for downstream analysis. In the first step, we amplified all samples with three loci . Samples that amplified for at least 2 loci were further amplified with the remaining of the final panel of 7 microsatellite loci. Samples that amplified for at least 5 (out of 7) loci were retained for further analysis.We used several lab precautions and analytical approaches to minimize errors that are characteristic of fecal DNA. We extracted DNA samples in small batches with a negative control in each set, used filter tips, and conducted extraction in a dedicated room that contained no previous bear PCR products. We used a modified multi-tubes approach , whereinID (probability of identity) and PID(sibs) values. PID and PID(sibs) values are commonly used measures to evaluate the power of the selected panel of loci to distinguish individuals [We measured genetic diversity by estimating the number of alleles per locus (Na), observed (Ho), and expected (He) heterozygosity in GIMLET. We conducted tests for deviations from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) in GENEPOP 4.0.10  with a Bividuals .ST values and 95% confidence intervals in FSTAT [ST and Euclidean geographic distances in GENEPOP. In addition, we conducted Mantel tests between each pair of individuals within each of the four tiger reserves in GenAlEx 6.5[To measure population-level differentiation, we measured pairwise Fin FSTAT . We usednAlEx 6.5.ST has been demonstrated to be a poor estimator of genetic differentiation that may have been caused by recent habitat fragmentation, because it has a lag time of about 200 generations [Ferations ,40. Indierations  package We used the Bayesian assignment program, STRUCTURE , to deteWe also used a spatially-explicit Bayesian analyses in TESS 2.3.1 . TESS usWe used the q-matrix from the STRUCTURE results to detect individuals of admixed ancestry, residents and migrants. We considered individuals with q values > 0.80 as residents of the area where they were sampled; those with q values from 0.2–0.8 as admixed individuals that could not be readily assigned as residents or migrants; and those with q values <0.2 as migrants .We used GENECLASS2  to examiFinally, we used BAYESASS1.3 to estimST (a measure of genetic differentiation).To test the hypothesis about correlation of forest connectivity and genetic differentiation, we calculated least-cost distance and Euclidian distance using the program Linkage Mapper . We usedOver a period of two years, we collected 190 fecal and 4 hair samples from wild sloth bears throughout the Satpura-Maikal landscape . This inWe did not detect any evidence of stuttering errors and large allele dropout in any of the 7 microsatellite loci using MICROCHECKER. Genotyping error rates  were low  . Two locID sibs 2.15E-03) from this pool, and there were three individuals that were recaptured twice using exact match criteria. The sample sizes across reserves were: 9 individuals from Kanha, 8 from Pench, 16 from Satpura, and 22 from Melghat . Fifty eight samples were retained for further analysis when we applied the criteria of amplification of at least 5 out of the total 7 loci. We were able to successfully amplify 80.78% of the total expected alleles. We identified 55 individuals . Pairwise FST values were significant between Kanha and Satpura, Pench and Satpura, and Pench and Melghat  on STRUCTURE results, we randomly reduced the number of individuals from Satpura and Melghat to make sample sizes similar across all four populations. In this analysis, we had the following sample sizes: Kanha (9), Pench (8), Satpura (9) and Melghat (8). We found that even after randomly removing individuals from Satpura and Melghat, we still detected K = 2 . This adThe analysis of q-matrix of STRUCTURE results revealed that eleven individuals (20%) were admixed , ten individuals (18%) were potential migrants, and 34 individuals (62%) were residents. GENECLASS detected five of these potential migrant individuals as first-generation migrants. One individual sampled in Kanha (assigned to Satpura), two individuals in Satpura (assigned to Kanha and Pench), and two individuals in Melghat (assigned to Satpura and Pench) were identified as being migrants .In the BAYESASS analysis, the highest and lowest proportions of non-migrants (both residents and admixed) were detected in Melghat  and Satpura , respectively. The highest proportion of migrants were from Melghat to Satpura (0.26) and the lowest proportion of migrants were from Pench to Melghat (0.01) .ST.To assess whether genetic differentiation and forest cover are correlated, we built resistance layers using a layer of forest cover and calculated Euclidian and least cost path distances. We found that neither Euclidian distance , nor least cost distance  was significantly correlated with FThis is the first study of population and conservation genetics of sloth bears. We confirmed the reliability of a panel of seven polymorphic microsatellite loci to monitor sloth bear populations using noninvasively collected samples. We also demonstrate its utility to study population genetic parameters and gene flow between sloth bear source populations in the Satpura-Maikal landscape in Central India.It has been documented that collecting fresh samples improves amplification success . HoweverDespite the challenges associated with amplification of noninvasive samples , we wereUrsus arctos)  [Ursus maritimus) (Ho = 0.68) [Ursus americanus)  [Differences in study design and types and numbers of markers hinder direct comparisons between studies that have examined levels of genetic variation in different species of ursids. However, our results suggest that the sloth bear population of central India has moderate levels  compared to estimates of population genetic diversity previously reported for Brown bears (3–8.312) , 24, 28, = 0.68)  and Amer.25–9.5) , 54.Panthera tigris) and leopards (Panthera pardus) in the Satpura-Maikal landscape [We used three different analytical approaches to detect genetic structure among sloth bear populations of Central India. These methods differ in their assumptions, and whether or not they include spatial information. All three analytical approaches indicate the presence of two genetic clusters. These two clusters are pairs of populations that are interconnected by existing corridors (Kanha-Pench and Satpura-Melghat). Previous studies on tigers  were confirmed as first-generation migrants and 20% of the individuals were of mixed ancestry, indicating admixture between the populations. These results show that sloth bears in this landscape are moving from one population to another and effectively breeding in the recipient population, thus maintaining genetic exchange and preventing further genetic subdivision in this landscape.We did not find a significant relation between forest cover and genetic distance. We think that this could be due to several factors: (1) Although least cost distances on an average are 21 km longer than Euclidian distances, the distances are highly correlated (R = 0.97 p = 0.04). The way this landscape is configured, it just so happens that Euclidian distance and least cost paths overlap to a certain degree. (2) Forest cover alone may not be a sufficient explanatory variable for genetic differentiation. Other factors such as food availability , and impact of human density, transportation networks and other infrastructure would be critical and ecologically meaningful for dispersing sloth bears.The best way to thoroughly establish the use and effectiveness of corridors by any species would be to combine genetic studies with movement tracking studies of multiple individuals to map their movement and effective dispersal through different landscape features. However, this is beyond the scope of our study, and our results using noninvasive genetic sampling provide strong evidence about the functionality of these corridors.Coal mining: The Central Indian Highlands are one of the major coal fields of India. There are several operational coal mines in Pench-Satpura corridor. In recent years, new mining leases were granted to mines proposed to be located inside this corridor. Mining activity has the potential to increase anthropogenic disturbance and reduce the connectivity through this important corridor .Widening of National Highway 7 (NH7): NH7 runs parallel to the eastern boundary of Pench tiger reserves (MP and Mh) for a length of 65 km. A stretch of 8.9 km of this highway intersects the Kanha-Pench corridor close to Pench tiger reserve (MP). The proposed widening of NH7 ,62 near Gauge conversion of railway line: A project to convert a 74.9 km stretch  of narrow-gauge railway line to broad-gauge is in progress. A 17.98 km long section of this railway line passes through forested area in the Kanha-Pench corridor and would impact 69.75 hectares of forest land . In addiTeak-monoculture: Multiple forest blocks adjacent to the tiger reserves and in the corridors in the Satpura-Maikal landscape have been demarcated for teak production. These monoculture blocks are devoid of any understory or fruiting trees ,67, and Fragmentation and loss of habitat is one of the most serious causes of population decline and species extinction globally , which aLynx rufus) and coyotes (Canis latrans) across a California freeway using genetic methods and radio-telemetry and found that although a few individuals of both species moved across the road, this movement was not enough to compensate for the restricted levels of gene flow caused by the freeway. Kerley et al. (2002) [Linear infrastructure features such as roads and railway lines have been shown to have severe adverse impacts on the behavior, movement, and gene flow of wildlife ,68. For . (2002)  revealedSome of these detrimental impacts can be mitigated by the use of specially designed structures such as overpasses and underpasses . A few sWe demonstrated that forest corridors in the Satpura-Maikal landscape are being used and are functional for movement and gene flow of sloth bears. However, no government policies are in place to safeguard these crucial areas of connectivity. Our study calls for immediate policy intervention for conservation planning to maintain linkages between protected areas in this landscape.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


STAT3 plays a pivotal role in the hematopoietic system, which constitutively activated by BCR–ABL via JAK and Erk/MAP-kinase pathways. Phospho-STAT3 was overexpressed in imatinib-resistant CML patients as relative to imatinib responsive ones. By activation of the STAT3 pathway, BCR–ABL can promote cell cycling, and inhibit differentiation and apoptosis. Ribosomal protein S27a (RPS27a) performs extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. RPS27a can promote proliferation, regulate cell cycle progression and inhibit apoptosis of leukemia cells. However, the relationship between STAT3 and RPS27a has not been reported. In this study, we detected a significantly increased expression of STAT3 and RPS27a in bone marrow samples from CML-AP/BP patients compared with those from CML-CP. In addition, we also demonstrated that it was a positive correlation between the level of STAT3 and that of RPS27a. Imatinib-resistant K562/G01 cells expressed significantly higher levels of STAT3 and RPS27a compared with those of K562 cells. RPS27a could be transactivated by p-STAT3 through the specific p-STAT3-binding site located nt −633 to −625 and −486 to −478 of the RPS27a gene promoter in a dose-dependent manner. The transactivated RPS27a could decrease the percentage of apoptotic CML cells induced by imatinib. And the effect of STAT3 overexpression could be counteracted by the p-STAT3 inhibitor WP1066 or RPS27a knockdown. These results suggest that drugs targeting STAT3/p-STAT3/RPS27a combining with TKI might represent a novel therapy strategy in patients with TKI-resistant CML. Chronic myelogenous leukemia (CML) is a common hematologic malignancy, characterized by the formation of Philadelphia (Ph) chromosome and BCR-ABL fusion gene . As a coRibosomal protein S27a (RPS27a) is one of two (the other one is L40) ribosomal proteins naturally synthesized as an ubiquitin (Ub) C-terminal extension protein . BesidesTo explore the mechanism of RPS27a up-regulation in the transition from CML-CP to CML-AP/BP, We hypothesized that some highly expressed proteins  in imatinib resistant CML cells contributed to the up-regulation of RPS27a gene , 22, 32.To explore the mechanism of RPS27a up-regulation in the transition from CML-CP to CML-AP/BP, We hypothesized that some highly expressed proteins  in imatinib resistant CML cells contributed to the up-regulation of RPS27a gene. The DNA sequence of 1.6-kb full-length human RPS27a gene (FL-RPS27a) was cloned into luciferase reporter constructs. The transcriptional regulation of RPS27a was investigated using the luciferase reporter assay after transiently transfecting cells with pCMV-P-gp, pCMV-AXL, pCMV-Hsp-70, pCMV-STAT3, pCMV-STAT5, pCMV-CIP2A, pCMV-BMI-1 and pCMV-ABCG2. There was a significant (about ten-fold) increase in the FL-RPS27a reporter gene activity in the presence of pCMV-STAT3 Figure . It implTo explore the possible role of STAT3 and RPS27a in CML cells, we first investigated the expression levels of STAT3 and RPS27a in CML-CP and CML-AP/BP. Relative quantification using qRT-PCR revealed a striking increase of STAT3 and RPS27a mRNA expression in bone marrow samples from CML-AP/BP patients than that from CML-CP patients Figure . And theTo explore the possible relationship of STAT3 and RPS27a, we investigated the changes of p-STAT3 and RPS27a expression after STAT3 was over-expressed in HEK293T cells. Relative quantification using qRT-PCR revealed a striking increase of RPS27a mRNA expression as STAT3 was overexpressed in HEK293T cells Figure . The oveAs shown in the above study, the endogenous expression of RPS27a could be up-regulated by p-STAT3, and on the other hand, there are putative p-STAT3-binding sites within the promoter region of RPS27a gene; there might be possible links between transactivation of RPS27a gene and p-STAT3 binding to its promoter. To this end, we generated a serial of RPS27a luciferase reporter gene constructs to analyze the trans-active effect of p-STAT3 on RPS27a. Of them, S934B containing two putative p-STAT3 protein-binding sites (nt −633 to −625 TTCCAGGAA and −486 to −478 TTCTAAGAA) and S594B containing one putative p-STAT3 protein-binding sites (nt −486 to −478 TTCTAAGAA) were fused to luciferase reporter vector pGL3-basic; whereas S441P containing one putative p-STAT3-binding site (nt −633 to −625 TTCCAGGAA) to pGL3-promoter vector Figure .in vivo-specific binding of p-STAT3 to promoter of the RPS27a gene.Each of these luciferase reporter gene constructs was co-transfected with pCMV5-STAT3 into HEK293 cells, respectively. The luciferase activity was analyzed by luminometer at 48 h after transfection. To demonstrate this ‘site-specificity’, Mu934B with mutated p-STAT3-binding site (nt −633 to −625 TGCCCGGGA and −486 to −478 TGCTCAGGA) was co-transfected with pCMV5-STAT3 into HEK293 cells. As shown in Figure As STAT3 and RPS27a were highly expressed in CML-AP/BP and the expression of RPS27a could be up-regulated by p-STAT3, the role of STAT3 and RPS27a in CML cells was elucidated by using K562 cells. We investigated the level of RPS27a, p-STAT3 and STAT3 after infection with pcDNA3.1-STAT3. As expected, stable infection experiments showed RPS27a, p-STAT3 and STAT3 could be significantly up-regulated in K562-STAT3 cells in mRNA and protein levels, compared with those of K562-con cells Figure . Then weTo confirm the effect of STAT3 overexpression in the regulation of cell apoptosis induced by imatinib of CML cells relies on the overexpression of p-STAT3. WP1066, one p-STAT3 inhibitor was applied to block the activity of p-STAT3 protein Figure . Then weTo confirm the effect of STAT3 overexpression in the regulation of cell apoptosis induced by imatinib of CML cells relies on the transactivation of RPS27a by p-STAT3. One RPS27a specific shRNA was applied to down-regulate RPS27a expression in K562-STAT3 cells. As expected, stable infection experiments showed RPS27a-shRNA could significantly down-regulate RPS27a expression in K562 -STAT3 cells in mRNA and protein levels Figure . Then weIn this study, we found that there was a striking increase of STAT3 and RPS27a mRNA expression in bone marrow samples from CML-AP/BP patients than that from CML-CP. And there was a positive correlation between the expression level of STAT3 and that of RPS27a. K562/G01 cells expressed significantly higher levels of STAT3 and RPS27a compared with those of K562 cells. RPS27a could be transactivated by p-STAT3 through the specific p-STAT3-binding sites located nt −633 to −625 and nt −486 to −478 in the RPS27a gene promoter in a dose-dependent manner. RPS27a transactivated by p-STAT3 could inhibit the percentage of apoptotic CML cells to imatinib.RPS27a can perform extra-ribosomal functions. RPS27a are overexpressed in mouse liver cancer and some human tumors –36. In pin vivo-specific binding of p-STAT3 to promoter of the RPS27a gene. These data suggested that the STAT3/p-STAT3/RPS27a signal pathway might participate the transition from CML-CP to CML-BP.STAT3 plays a pivotal role in the hematopoietic system . The levIn previous study, we found that RPS27a promotes inhibits the effect of imatinib on apoptosis of CML cell lines . In the In conclusion, STAT3 and RPS27a mRNA expression were up-regulated in the transition from CML-CP to CML-AP/BP. And there was a positive correlation between the expression level of STAT3 and that of RPS27a. RPS27a could be transactivated by p-STAT3 through the specific p-STAT3-binding site located nt −633 to −625 and −486 to −478 in the RPS27a gene promoter in a dose-dependent manner. RPS27a transactivated by p-STAT3 could inhibit chemosensitivity of CML cells to imatinib. It appears that drugs targeting STAT3/p-STAT3/RPS27a combining with TKI might represent a novel therapy strategy in some patients with TKI resistant CML.Bone marrow samples were obtained from 20 CML-CP patients and 15 CML-AP/BP enrolled in Department of Hematology, Shanghai Tenth People's Hospital, Tongji University School of Medicine. Two bone marrow samples were obtained from respective CP and BP of two CML patients . All samples were collected under informed consent of the subjects. The diagnosis and leukemia classification were based on 2008 World Health Organization criteria.2 in air. Imatinib at concentration of 1 μM was added to the culture system of K562/G01 cells to maintain resistance activity.K562, HEK293T, HEK293 and K562/G01 cell lines were maintained in our laboratory. The HEK293T and HEK293 cell lines were cultivated in Dulbecco's modified eagle medium and K562 and K562/G01 cell lines in RPMI 1640 medium containing 10% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% COTotal RNA was extracted using Trizol Reagent treated with DNase I and 2 μg RNA was reverse-transcribed using Superscript II RT following the manufacturer's instructions . Primers for qRT-PCR were designed using Primer premier software 5.0. Human GAPDH primers used as an internal control were 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse). Human RPS27a primers were 5′-AGAAGAAGTCTTAC ACCACTCCC-3′ (forward) and 5′-TGCCATAAACACCC CAGC-3′ (reverse). Human STAT3 primers were 5′-CAGT TTCTTCAGAGCAGGTA-3′ (forward) and 5′-CTTGACT CTTGAGGGTTTT-3′ (reverse). The qRT-PCR products were 226 bp, 158 bp and 286 bp, respectively. The qRT-PCR was performed with SYBR Green PCR kit  following the manufacturer's instructions on the ABI PRISM7500 real-time PCR system. Thermal cycling conditions were 95°C for 5 min, followed by 40 cycles of 5 s at 95°C, and 34 s at 60°C. The qRT-PCR reactions were performed in a total volume of 20 μl, containing 2 μl of sample cDNA, 0.2 μM of each primer.3VO4, 0.1 mM PMSF and 2 mg/ml Aprotinin). Protein concentration was determined by the BCA assay . The whole-cell lysates were heat-denatured at 95°C for 5 min before run on 10% SDS- PAGE. After SDS-PAGE, the proteins were electro-transferred onto nitrocellulose membranes, blotted with each primary antibody, incubated in secondary antibody and then detected with enhanced chemi-luminescence detection reagent .Total protein was extracted using RIPA lysis buffer  was from nt −1644 to −7 of the promoter sequence of RPS27a. The DNA fragment was cloned from the total DNA of K562 cells by PCR. The primers were 5′-AGCCAAGATCTGCCAAGGTA-3′ (forward) and 5′-CACTCCTTAGTGTGACACGCTTT-3′ (reverse). After transfected by calcium phosphate-mediated precipitation for 48 h, HEK-293 cells were collected for assays of β-galactosidase and luciferase activity according to the Dual-Luciferase Reporter 1000 Assay System Technical Manual  by luminometer . The following combinations of plasmids were used: 750 ng S934B, Mu934B, S594B or S441P reporter plasmids with different dosage of pCMV5-STAT3 and with 750 ng β-galactosidase plasmids.Chromatin immunoprecipitation analysis was performed with sonication method. The purified chromatin from K562/G01 cells was immunoprecipitated with anti-p-STAT3 antibody (Abcam). Anti-STAT3 antibody (Abcam), immunoglobulin G isotype and H2O were used as control. The eluted DNA was purified, and used as PCR template to amplify the promoter sequences between nt −670 and −467 containing putative p-STAT3-binding site at nt −633 to −625 and nt −486 to −478 of RPS27a promoter with the following primers: 5′-GAGCGAAATTCCGTCTC-3′ (forward) and 5′-TGACATTCAGCCTCTGC-3′ (reverse). The PCR products were 184 bp.3(PO4)2 precipitation. Lentivirus supernatants were harvested 48 h after transfection, and used to infect K562-STAT3 cells (termed as K562-STAT3-RPS27a-sh and K562-STAT3-RPS27a-scr). Infected cells were selected by puromycin 7 days after infection for at least 2 weeks to obtain stable clones. Inhibition of RPS27a expression was measured by qRT-PCR as well as by Western blot using a rabbit anti-RPS27a monoclonal antibody .The cDNA sequence of RPS27a was obtained from GenBank (NM_002954.5). A RPS27a-specific targeting sequence was designed with the software from Ambion. The target sequence for RPS27a was 5′-TTAGTCGCCTTCGTCGAGA-3′. The shRNA expressing plasmid specifically targeting RPS27a was cloned into pLKO-1 vector to obtain pLKO-shRNA construct. A scramble shRNA sequence (AATAGACTCAGCAAATGCG) with no homology to any human gene was used as a negative control (scr). For the production of lentivirus, HEK293T cells were co-transfected with pLKO-shRNA or pLKO-scr and pCMV, pMDG by Ca3 cells per well were seeded into 96-well plates in 100 μl volume and grown in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C. Cells were cultured for 48 h and 72 h, respectively. After indicated time of incubation, 10 μl of MTT reagent was added. Then the cells were incubated at 37°C for an additional 4 h, and 100 μl of 10% sodium dodecyl sulfate-HCl 0.1 N was added to each well. The mixture was incubated at 37°C overnight and then the absorbance of formazan product was measured by Versa Max tunable microplate reader  at the wavelength of 546 nm. The in vitro cytotoxicity of K562 and K562-STAT3 cells and their transfectants were assessed by MTT assays.5 × 10Phosphatidylserine externalization was analyzed with Annexin V-Alexa Fluor 647-A/PI Apoptosis Analysis Kit by a FACS Calibur flow cytometer  for cell apoptosis. Apoptosis was quantified as the percentage of Annexin V positive cells.P < 0.05 was deemed statistically significant.All experiments were conducted at least three times and data were presented as mean ± SD. Statistical analysis was performed with the SPSS software package .


This paper presents for the first time a comparison of the genetic consequences of two different types of speciation in plants of an oceanic island. Genetic data, using two different DNA methods, were obtained from more than 4,000 plants from the two major islands of the Juan Fernández Archipelago (Chile). Results show that some immigrant populations undergo major splitting events and harbor limited genetic diversity within each evolving line. In contrast, other immigrant populations establish and enlarge, but they never split, hence accumulating higher levels of genetic diversity. Drimys (Winteraceae), Myrceugenia (Myrtaceae), Rhaphithamnus (Verbenaceae), Robinsonia  and Erigeron . The results indicate that species originating anagenetically show high levels of genetic variation within the island population and no geographic genetic partitioning. This contrasts with cladogenetic species that show less genetic diversity within and among populations. Species that have been derived anagenetically on the younger island (1–2 Ma) contain less genetic variation than those that have anagenetically speciated on the older island (4 Ma). Genetic distinctness among cladogenetically derived species on the older island is greater than among similarly derived species on the younger island. An important point is that the total genetic variation within each genus analysed is comparable, regardless of whether adaptive divergence occurs.Adaptive radiation is a common mode of speciation among plants endemic to oceanic islands. This pattern is one of cladogenesis, or splitting of the founder population, into diverse lineages in divergent habitats. In contrast, endemic species have also evolved primarily by simple transformations from progenitors in source regions. This is anagenesis, whereby the founding population changes genetically and morphologically over time primarily through mutation and recombination. Gene flow among populations is maintained in a homogeneous environment with no splitting events. Genetic consequences of these modes of speciation have been examined in the Juan Fernández Archipelago, which contains two principal islands of differing geological ages. This article summarizes population genetic results  from examination of 15 endemic species, involving 1716 and 1870 individuals in 162 and 163 populations  in the following genera:  Oceanic islands have long stimulated biologists to investigate patterns and processes of evolution e.g. . These iAeonium , another process, anagenesis Fig. , has recEchium (Boraginaceae) of the Canary Islands, showing very little nucleotide divergence even though the morphological variation is striking. Likewise, Some studies have been published on the genetic consequences of cladogenesis in endemic plants of different archipelagos. Dystaenia takesimana and D. ibukiensis, respectively, and the results showed high levels of genetic variation within D. takesimana in comparison with D. ibukiensis. Similar results have been obtained in assessing the origin of Acer takesimensis and A. okomotoanum , which occur on both islands of the archipelago. Investigations have also been completed on Myrceugenia  genus that has been investigated is Drimys, Myrceugenia, Rhaphithamnus, Robinsonia and Erigeron; (ii) compare and contrast differences in genetic diversity in groups that have undergone anagenetic or cladogenetic speciation and (iii) discuss the importance of considering modes of speciation for understanding levels of genetic diversity within endemic species of oceanic archipelagos.The purposes of this article are to (i) summarize published data from AFLP and SSR investigations on endemic species of the genera The data summarized here Table  provide Briefly, the following approaches were used for AFLPs. Four or six selective primer combinations were chosen. Numerous (24–85) primer trials were run with each genus to determine the best combination of primers for good resolution of individuals and populations. Data were obtained on an automated DNA sequencer . Scoring was done using GeneMarker ver. 1.85 . For analysis of AFLP data, the programs Arlequin 3.5.1.2 , R-ScripHO), expected proportion of heterozygotes (HE), number of alleles per locus (NA), inbreeding coefficient (FIS) and allelic richness standardized by five individuals (AR5).For SSRs, NGS methods , Micro-Ct-test  and shown in Table HO and HE, a square-root transformation was applied. The overall patterns of higher genetic diversities in Robinson Crusoe Island (old) than Alejandro Selkirk Island (new) were also examined in the same way. The effects of two factors (speciation mode and island) and their interaction were analysed in a two-way ANOVA in R version 3.0.0  in populations on the older island than on the younger island except for estimates of SSRs in R. venustus . Although one cannot place an absolute time on this divergence, it is the lowest level among any pair of species in the genus, which correlates well with the youthful geological age of Alejandro Selkirk Island. Genetic variation in R. masafuerae is much lower from AFLP data than in R. evenia from Robinson Crusoe . Robinsonia has adaptively radiated on Robinson Crusoe Island during the past 4 million years  and Erigeron has done so on Alejandro Selkirk Island in the past 1–2 million years.Two of the largest genera of the archipelago are Robinsonia is the second largest genus in the archipelago. The largest is Dendroseris, also of Asteraceae but from still another tribe (Cichorieae). This latter genus is of interest as it has derived cladogenetically on the older island with three independent dispersals to the younger island and three anagenetic speciations there  on Alejandro Selkirk Island. The results from AFLP data are shown in Fig. Robinsonia are very distinct genetically. Divergence has obviously taken place during adaptive radiation and also during a maximum time available of 4 million years. The species R. gayana, R. thurifera and R. saxatilis form an evolutionary complex, which taxonomically has been regarded as sect. Robinsonia  presumably arrived directly to the younger island ; its close relative, E. stuessyi, is also found on rocky ledges but residing inside the cool and deep ravines. Erigeron fernandezianus occurs in a broad altitudinal range (100–1200 m), and it inhabits mainly rocky areas in middle elevation plains, quebradas and ridges. This species also occurs on the older island, but it is found there in many plant communities and especially in disturbed sites. It appears, therefore, to be an example of back migration from the younger to the older island  and inter simple sequence repeats (ISSRs). Isozymes have been analysed in Hes = 0.065). Higher levels of diversity were seen in larger populations or in many small populations and also in outcrossing species in contrast to selfers. Of relevance for comparisons to the present study, isozymes have been analysed from four species of Robinsonia and in M. fernandeziana, E. fernandezianus and R. venustus. It is difficult to compare the results of the isozymes because they provide less detailed genetic information than from AFLPs and SSRs. Isozyme studies on the endemic Lactoris fernandezianus, for example Dendroseris showed greater resolution of relationships from the latter. The isozyme data for the four cladogenetically derived species of Robinsonia show higher levels of genetic variation than in the anagenetic R. venustus , incursions into the native forest must have taken place and some plants destroyed. It is known that two species of Robinsonia, both on Robinson Crusoe Island, are now extinct  grant (P21723-B16) to T.F.S. and a Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship for Research Abroad (grant 526) to Ko.T.This work was supported by an FWF (Ko.T. conceived the idea behind the article; all authors participated in the field work except G.K. and Ka.T.; J.N., P.L.-S., G.K. and Ko.T. completed the laboratory work; J.N. coordinated the NGS data acquisition; T.F.S. and Ko.T wrote the initial draft and all authors contributed to subsequent drafts and offered comments for improvement.None declared.


Molecular characterization information on genetic diversity, population structure and genetic relationships provided by this research will help maize breeders to better understand how to utilize the current CML collection.CIMMYT maize inbred lines (CMLs) have been widely used all over the world and have contributed greatly to both tropical and temperate maize improvement. Genetic diversity and population structure of the current CML collection and of six temperate inbred lines were assessed and relationships among all lines were determined with genotyping-by-sequencing SNPs. Results indicated that: (1) wider genetic distance and low kinship coefficients among most pairs of lines reflected the uniqueness of most lines in the current CML collection; (2) the population structure and genetic divergence between the Temperate subgroup and Tropical subgroups were clear; three major environmental adaptation groups  were clearly present in the current CML collection; (3) the genetic diversity of the three Tropical subgroups was similar and greater than that of the Temperate subgroup; the average genetic distance between the Temperate and Tropical subgroups was greater than among Tropical subgroups; and (4) heterotic patterns in each environmental adaptation group estimated using GBS SNPs were only partially consistent with patterns estimated based on combining ability tests and pedigree information. Combining current heterotic information based on combining ability tests and the genetic relationships inferred from molecular marker analyses may be the best strategy to define heterotic groups for future tropical maize improvement. Information resulting from this research will help breeders to better understand how to utilize all the CMLs to select parental lines, replace testers, assign heterotic groups and create a core set of breeding germplasm. Molecular characterization of genetic diversity, population structure and genetic relationships among elite breeding materials within a given set of maize germplasm is useful for understanding how to use the assembled germplasm for further improvement, such as selecting parental lines, assigning heterotic groups and creating a core set of germplasm. Maize germplasm can be divided into two major groups—i.e., temperate and tropical germplasm—based on environmental characteristics, particularly day length. The International Maize and Wheat Improvement Center (CIMMYT) focuses mainly on tropical maize germplasm improvement. CIMMYT maize inbred lines (CMLs) are carefully selected for good general combining ability and a significant number of value-added traits such as drought tolerance, nitrogen use efficiency, resistance to disease and insect pests, and grain nutritional quality. Generally, two heterotic groups were classified within CMLs, i.e., the “A” group is dent kernel type and “B” group is flint kernel type. CIMMYT CMLs are widely used by public and private institutions worldwide, especially in developing countries , amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs) or single nucleotide polymorphisms (SNPs) , a next-generation sequencing (NGS) technology, is a high-throughput, multiplex and short-read sequencing approach that reduces genome complexity via restriction enzymes and generates high-density genome-wide markers (~1 million) at a low per sample cost by tagging randomly shared DNA fragments from different samples with unique, short DNA sequences (barcodes) and pooling samples into a single sequencing channel  understand the genetic diversity of a tropical maize collection comprising all CMLs; (2) investigate the population structure and heterotic patterns of this collection; (3) estimate the genetic relationships among the most important CMLs; and (4) assess how GBS could be utilized to perform genetic diversity analyses in tropical maize.http://hdl.handle.net/11529/10246. All the released CMLs are publicly available to anyone in the world and could be requested through the CIMMYT Maize Germplasm Bank online seed ordering site: http://www.cimmyt.org/en/what-we-do/germplasm-and-seed/obtainseed.A panel of 538 CMLs and 6 temperate inbred lines was used for molecular characterization analysis. CML250 was not included due to missing sample issues. Basic information on all of the lines including their environmental adaptation, number of lines in each geographic subset, grain color, grain texture and testers in each adaptation group is summarized in Table Based on geographic information and environmental adaptation, 538 CMLs were classified into seven subsets: (1) Mexico Lowland: 241 lines developed in Mexico; (2) Africa Lowland: 22 lines developed in Kenya and Zimbabwe; (3) Asia Lowland: 20 lines developed in Thailand; (4) Mexico Subtropical: 145 lines developed in Mexico; (5) Africa Mid-altitude: 58 lines developed in Kenya and Zimbabwe; (6) South America: 22 lines developed in Colombia; and (7) Highland Tropical: 30 lines developed in Mexico. For convenience, we refer to the Mexico Lowland, Africa Lowland and Asia Lowland subsets as the Lowland Tropical subgroup; the Mexico Subtropical, Africa Mid-altitude and South America subsets as the Subtropical/Mid-altitude subgroup; and the Highland Tropical subset as the Highland Tropical subgroup, since they are from different CIMMYT maize breeding programs targeting similar environments. Six temperate maize inbred lines  were referred to as the Temperate subgroup and used as a reference to assess the genetic divergence between temperate and tropical maize.ApeK1. GBS libraries were constructed in 96-plex and sequenced on Illumina HiSeq 2000. SNP calling was performed using TASSEL-GBS pipeline with B73 as the reference genome . This collection includes genotypes of more than 60,000 maize samples. In this study, we focused on the subset of 539 genotyped CMLs, and un-imputed raw GBS data were used for further genetic characterization analyses. Initially, 955,690 SNPs evenly distributed on maize chromosomes were called for each line; 955,120 of them were assigned to chromosomes 1–10, and 570 of them could not be anchored to any of the 10 maize chromosomes. A filtered subset of 362,008 SNPs with minor allele frequency (MAF) greater than 0.05 was selected for genetic characterization analyses in this study. For all the maize lines tested in this study, un-imputed 955,690 SNP dataset and filtered 362,008 SNP dataset are publicly available on the CIMMYT Research Data Repository website: http://data.cimmyt.org/dvn.For all the maize lines tested in this study, leaf samples bulked from 15 plants of each line were used for DNA extraction with a CTAB procedure  between vectors of SNP alleles was used to assess the level of LD decay on each chromosome, and average LD decay distance across ten chromosomes with r2 = 0.1 was used to measure the difference of LD decay distance between the entire panel and other subgroups  between SNPs on each chromosome was measured with 362,008 SNPs in TASSEL 5.0. Squared Pearson correlation coefficient  was used to determine the number of clusters  to assess the genetic relationship among all 544 maize inbred lines, and missing data in PCA were imputed to mean allele frequency. The first two principal components were illustrated for the entire panel and for each environmental adaptation.Principal component analysis (PCA) based on 362,008 SNPs was performed with the R software  is provided in Table The distribution of pairwise relative kinship for 544 maize inbred lines estimated with 362,008 SNPs is shown in Fig. r2 = 0.1 was 3.76 kb. In Lowland Tropical subgroup, Subtropical/Mid-Altitude subgroup and Highland Tropical subgroup, the average LD decay distances over all ten chromosomes with r2 = 0.1 were 4.31, 3.12 and 3.56 kb, respectively  based on environmental adaptations and CIMMYT maize breeding history. According to information of environmental adaptation and pedigree, the Lowland Tropical subgroup consists of 283 lines derived mainly from the Mexico Lowland, Asia Lowland and Africa Lowland environmental adaptations. The Subtropical/Mid-altitude subgroup consists of 225 lines derived mainly from the Mexico Subtropical, Africa Mid-altitude and South America environmental adaptations. The number of lines in the Lowland Tropical and in the Subtropical/Mid-altitude subgroups is similar to the environmental adaptation and pedigree information. However, the Lowland Tropical subgroup seems to have many more inbred lines than the Subtropical/Mid-altitude subgroup in Fig. Results of population structure analysis of all 538 CMLs for PCA results based on 362,008 SNPs clearly distinguished three major subgroups among all CMLs: Lowland Tropical, Subtropical/Mid-altitude and Highland Tropical Fig. . This coThe genetic distance among different subgroups was estimated with the 362,008 SNPs Table ; resultsGenetic characterization of each subgroup was quantified by calculating the average values of PIC, heterozygosity rate and gene diversity of all the lines within each subgroup Table . The PICTHe UPGMA tree results based on Rogers’  genetic GBS is a low coverage sequencing technology which results in a very high missing rate, for example, in both the 955,120 and 362,008 SNP datasets it is above 50 %, and high levels of missing data can be a problem for downstream analysis like association mapping. Generally, imputation of missing data is not necessary for genetic diversity analysis. Imputation uses information from the other haplotypes to fill the missing data gaps, so basically it uses sample relatedness to create the new dataset. Using imputed data to analyze sample relatedness could bias results, showing close haplotypes to be closer than they really are and vice versa. Additionally, more layers of error to the genotyping data happened on the SNPs toward regions of the genome with more chances of getting sequenced  and also regions with more chances of getting imputed  during SNP calling and imputation processes. It has been shown that even with high levels of missing rate, the GBS data generated without imputation can be used for genetic diversity and population structure analysis in the temperate maize diversity panel  the genotyping error of GBS in temperate maize has been reported as 0.42 % based on the data from NAM (nested association mapping) populations, where few tropical materials were included  have heterotic information estimated based on pedigree information and combining ability tests through diallel and line-by-tester analyses. Since only a limited number of lines can be included in each combining ability test experiment, it is not possible to estimate the heterotic group and genetic relatedness of all maize lines in the current CML collection via one general combining ability test. Molecular marker analyses provide an alternative approach for large-scale genetic diversity characterization within a given germplasm collection. However, it has been reported several times that the heterotic patterns in tropical maize collections are still not clear compared with temperate maize, and the heterotic patterns estimated based on molecular markers are not fully consistent with those estimated based on combining ability tests and pedigree information (Lu et al. The creation of heterotic groups in maize is based on long-term selection. The development of heterotic groups in temperate maize started around 100 years ago, but heterotic group development work in tropical maize at CIMMYT began only three decades ago, in the mid-1980s. Most CMLs were derived from broad germplasm pools, populations and open-pollinated varieties; only more recently released CMLs were developed from bi-parental crosses using the pedigree breeding method. So it is easy to understand why heterotic patterns in tropical maize are still not clear. But we also found that most of the lines from heterotic group A and heterotic group B tend to cluster together in the Lowland Tropical and Subtropical/Mid-altitude subgroups, respectively. This suggests that short-term selection for hybrid performance has contributed to classifying tropical maize heterotic patterns at CIMMYT. Therefore, combining the current heterotic information based on combining ability tests and the genetic relationships inferred from molecular marker analyses may be the best strategy to define heterotic groups for future tropical maize improvement. The results of this research will also help breeders to understand how to utilize all the CMLs in other breeding activities, such as selecting parental lines, replacing appropriate testers, assigning heterotic groups and creating a core set of germplasm.


Neovison vison) to determine whether the removal of this predator on the Koster Islands archipelago and the nearby Swedish mainland affected genetic variation over six consecutive years of mink culling by trappers as part of a population control programme. We found that on Koster Islands allelic richness decreased (from on average 4.53 to 3.55), genetic structuring increased, and effective population size did not change. In contrast, the mink population from the Swedish coast showed no changes in genetic diversity or structure, suggesting the stability of this population over 6 years of culling. Effective population size did not change over time but was higher on the coast than on the islands across all years. Migration rates from the islands to the coast were almost two times higher than from the coast to the islands. Most migrants leaving the coast were localised on the southern edge of the archipelago, as expected from the direction of the sea current between the two sites. Genetic monitoring provided valuable information on temporal changes in the population of American mink suggesting that this approach can be used to evaluate and improve control programmes of invasive vertebrates.Eradication and population reductions are often used to mitigate the negative impacts of non-native invasive species on native biodiversity. However, monitoring the effectiveness of non-native species control programmes is necessary to evaluate the efficacy of these measures. Genetic monitoring could provide valuable insights into temporal changes in demographic, ecological, and evolutionary processes in invasive populations being subject to control programmes. Such programmes should cause a decrease in effective population size and/or in genetic diversity of the targeted non-native species and an increase in population genetic structuring over time. We used microsatellite DNA data from American mink ( Ne, due to the increased rate of genetic drift and/or changes in gene flow among demes, or due to a decrease in fitness by selectively removing individuals with specific phenotypic traits from a population [Harvesting of animals in the wild, especially when intense, may lead to the direct extinction of a single population or even a whole species . Besidespulation . These ppulation . Furtherpulation , 4, 5. Apulation , 6. A mapulation .vs eradication of invasive non-native species) they relate to the same ecological theory, suggesting that overexploitation first poses genetic changes that often reduce recovery rates and further increases the extinction risk of the population [In the case of invasive species, negative genetic and demographic changes in populations may be desired and may provide information that will help to reduce population sizes or even to eradicate particular populations from the wild. As the number of introduced non-native species increases  there ispulation .et al. [As a tool for assessing the outcomes of the management of harvested species, Schwartz et al.  proposedet al. , 19. Usiet al. . To moniet al. .Effective management programmes geared toward reducing the population size of an invasive species must also focus on defining management units –23. To uNeovison vison) is an invasive species that has an impact on native biodiversity and ecosystem functioning, by affecting the populations of both their prey, and their competitors [Arvicola terrestris), coot (Fulica atra) and grebes (Podiceps spp.) has been observed on the mainland, but the impact is especially severe on sea islands where American mink reduces the number of seabirds in breeding colonies [The American mink  assess the impact of harvesting on allelic richness, heterozygosity and effective population size in consecutive years; (2) analyse the genetic structure of American mink inhabiting the mainland and islands to assess management units; (3) evaluate gene flow from the mainland to the islands to estimate the number of individuals re-colonising the islands.All procedures involving animals in this study fulfilled the ethical requirement stated by the European directive and Swedish legislation about animals in research. DNA samples were acquired post mortem from invasive American mink killed by licensed hunters  to prevent predation on ground nesting birds in protected areas. Animals were shot during the regular hunting season under the rules of the Swedish hunting law and the DNA-samples were given to us courtesy of the local hunters. Thus, no animals were killed specifically for this study.2 and consists of 800 small islands and islets with a total area of 8 km2. Most of the park is situated west of a deep water trench. The area also includes two main islands not included in the national park, which have a total area of about 12 km2 and are populated by some 300 persons. Common Eider (Somateria mollissima), Arctic Skua (Stercorarius parasiticus), Arctic Tern (Sterna paradisaea), Black Guillemot (Cepphus grille), Eurasian Oystercatcher  and several species of gulls (Larus spp.) are examples of marine birds breeding in the park. The presence of American mink is recognised as a major problem for seabird nesting, and the management plan includes an annual hunt of the species to reduce its number.To analyse the importance of culling on genetic diversity and gene flow in the American mink, we selected three sites in eastern Skagerrak: (1) a group of islands located 3–4 km from the coast with the shortest distance between islands and coast being 1.6 km (Koster Islands–KI) , (2) a mainland site (North coast–NC) located ca. 3 km from the Koster Islands, and (3) another mainland site (South coast–SC) located 30–100 km south of the islands . The samA total of 205 American mink tissue samples were collected from the islands and coastal areas between 2006 and 2011 . The majFIS) were calculated using GenePop on the Web version 4.2 [A), mean number of private alleles (Aprivate), observed heterozygosity (HO) and unbiased expected heterozygosity (uHE) using FSTAT 2.9.3 [Ar) using FSTAT.Evaluations of the presence of null alleles were performed using MicroChecker version 2.2.3 . Loci thsion 4.2  and BonfAT 2.9.3  and GenAAT 2.9.3 . The meaFST levels among predefined populations using FSTAT, and the determination of the recently developed alternative measure of genetic differentiation Dest [We used a range of different analytical approaches for identifying genetic differentiation across samples of American mink. Population genetic structure was first detected by the determination of ion Dest  using thion Dest . Second,K (ΔK) was used to find the most likely K [K), ranging from 1 to 7, were calculated from 10 independent runs to establish consistency. This method usually detects only the uppermost level of genetic structure [The genetic structure of American mink was further assessed using individual based analyses with the software STRUCTURE 2.2 , and a dlikely K . For eactructure , and in find.clusters function in adegenet 3.1.9, an R package dedicated to the multivariate analysis of genetic markers [et al. [dapc function in adegenet retaining a conservative seven PCs.Finally, cryptic genetic structures were analysed using a discriminant analysis of principal components . DAPC p markers . In thes [et al. . RetainiWe assessed the degree of dispersal (number of migrants) between sites using two different methods. First, we estimated the proportion of individuals with membership q≥0.8 in the first level of the structure analysis conducted with STRUCTURE (see above). Second, we estimated first generation migrants by genetic assignment testing of an individual to a population other than where it was actually collected, implemented in GeneClass2 version 2.0; ). We conassign) calculated by BIMR to extract parameter estimates. Density functions were analysed and the mode (point estimate) and 95% highest posterior density interval (HPDI) were noted.We also estimated current rates of gene flow between the sites using a Bayesian MCMC method that relaxes the Hardy-Weinberg equilibrium assumption by using population-specific inbreeding coefficients, implemented in BIMR 1.0 . Ten repNe) for each of the three geographic sites. The samples of mink were pooled into two culling season groups to get reasonable samples sizes. Effective population size is a crucial parameter in the management and eradication of invasive species because of its influence on population viability and the ability to predict extinction risk [The linkage disequilibrium (LD) and molecular coancestry (MC) approaches  were usion risk . The linion risk . As suggion risk , alleles2 of island in the KI area per hunting season. Males were culled at a higher proportion than females at all sites: on KI 59.5% of culled mink were males, NC– 57.8% and SC 73.1%.During eleven years (2001–2012) two hunters removed 260 mink from KI and 82 from the NC site. These hunters removed from 9 to 53 (on average 23.6) mink per hunting season on KI and a maximum of 17 (on average 7.5) mink from the NC site . An addiA) per locus within the sampling sites and years ranged from 4.1 to 6.3, the mean number of private alleles (Aprivate) from zero to 0.6 and the allelic richness (Ar) from 3.3 to 4.3 (A) and allelic richness (Ar) decreased significantly in consecutive years of mink eradication from 6.3 in 2006 to 4.1 in 2010 and from 4.5 to 3.5 respectively  was found in one locus (Mvi1302), which was subsequently excluded from further analysis. Fourteen of 2090 pairwise locus exact tests of linkage disequilibrium were significant after Bonferroni correction (P < 0.00002), but these were scattered randomly across locus pairs. All 20 microsatellite loci were polymorphic and overall a total of 166 alleles were found, with an overall mean of 8.3 (SE±0.45). The total number of alleles per locus ranged from 5 (Mvis002) to 12 (Mvi114 and Mvis099). The mean number of alleles . At the NC site both indices were similar among years and similar to SC . Three of the 11 temporal sampling sites  showed significant deviations from Hardy–Weinberg expectations after Bonferroni correction. In all instances this was due to a deficiency of heterozygote genotypes. Single-locus Hardy–Weinberg equilibrium tests showed that heterozygote deficits were attributable to the locus Mvis586 in the NI2006 sampling sites, locus Mvi114 in the NI2008 sampling sites, and locus Mvi4058 in the SC sampling site.All sampling sites showed intermediate values of heterozygosity: Hectively . In the FST values (0.031–0.116) were observed from comparisons between KI and both coast sites than between years within sites (vs coast) only 3 of 30 comparisons indicated no significant divergence after sequential Bonferroni correction, suggesting that gene flow restriction occurs between the islands and the coast. Differentiation was the highest between KI and SC (0.077–0.116) and moderate but significant differentiation was also observed between NC and SC (0.065–0.092). In the mink population that inhabits KI, the differentiation also increased between consecutive years of culling, from 0.003 between 2006 and 2007, to 0.030 between 2006 and 2011, suggesting genetic drift at this site  was used to assess the distribution of genetic variation at temporal and spatial scales. AMOVA indicated that only 1.5% of overall variance was attributable to differences among years within sites, with 6.8% of variance attributable to differences between the sites .The clustering approach used by STRUCTURE to determine the number of genetic groups across the entire data set identified two hierarchical levels of subdivision. Initial partitioning of the data indicated the presence of two clusters ; S2 Fig.The second round of STRUCTURE analysis for the assignment of individuals to cluster 2 split mink from both coast sites. The individuals from the NC were clearly grouped into one cluster (2a), and separated from individuals from the SC, which were grouped into another cluster (2b) . For indSimilar to analyses in STRUCTURE, the DAPC clustering algorithm based on BIC suggested five distinct clusters . The twoTwo different methods indicated recent migrants between sites. Based on STRUCTURE assignment tests at the first level, 6 mink (4.5%) sampled from KI were assigned to the NC group, whereas 4 mink (8.7%) from NC were assigned to a group of mink from KI, with thresholds q>0.8 . At the Dassign) indicated that the mean migration rate between the three sites ranged from almost no migration into SC from the two other sites, to as high as 14.9% from KI to NC  of 29.1 in KI  and 44.0 at NC   was indicated. The estimation of Ne based on the molecular coancestry method was from two to five times lower in comparison to the linkage disequilibrium method, but the proportion of Ne between sites was similar: Ne was highest at SC, moderate at NC and lowest at KI (The effective population size was lower in KI than at the two other sites. Over the entire sampling period (2006–2011), the linkage disequilibrium method gave an average effective population size (.6–58.2) . Based ost at KI .In this study we demonstrate that genetic monitoring can be used to evaluate the effectiveness of culling of invasive American mink. We used temporal microsatellite DNA data to test the hypothesis that culling mink from small islands has altered genetic diversity and effective population size. In consecutive years of mink culling on Koster Islands the allelic diversity decreased, genetic structuring in a temporal and spatial scale increased, but the effective population size did not change. In contrast, in both mainland populations, no temporal changes in genetic diversity and structuring were observed, suggesting genetic stability of these populations over six years of culling. Across all years, effective population size was higher at the mainland sites than on the Koster Islands.The eradication or population control of non-native invasive species has been carried out in many different localities on several continents , 10. In Leopardus pardalis) or red squirrel (Tamiasciurus hudsonicus) [Mustela erminea) population in New Zealand, found that the genetic diversity remained relatively stable after six years of removal [Overall, the genetic diversity of introduced non-native species is related to propagule pressure: mainly it is related to the number of introduction events and the number of introduced individuals , 62–64, sonicus) , 70. The removal . Given tPanthera tigris), Florida panthers (Puma concolor), chimpanzees (Pan troglodytes) or harbour seals (Phoca vitulina), [The causal role of genetic diversity in population extinction or decline is not well documented and is still controversial (see for example ), but retulina), –78). Simtulina), , 80). ThFST values significantly greater than zero. Second, assignment tests placed more than 85% of individuals into their sampled population. Third, Bayesian clustering methods implemented in STRUCTURE, with no a priori information on an individual’s origin, grouped samples according to their sites of origin. Similar results were obtained by DAPC analysis, which also revealed the presence of five clusters in the study sites. Interestingly, the removal of mink increased differentiation between the KI and NC sites. The pairwise FST values were lowest when we compared mink individuals sampled on KI in 2006 to mink sampled at NC in the next years. The FST values increased and were highest when we compared mink from KI sampled in 2011 to mink sampled at NC in all other years. The observed increase in genetic structuring was probably affected by the reduction of the number of migrants from both sites. Furthermore, we also found weak genetic structuring within KI, in different groups of individuals in the eastern and western part of the island archipelago. A total of five clusters of American mink were identified  which partially reflected their distribution across the sites, but this also suggested that there were other factors shaping mink genetic structure (most probably history of island colonisation) as individuals from KI were grouped into three clusters in a west-east gradient.An understanding of the population genetic structure in management areas is necessary to predict the potential effect of culling on genetic subdivision, and also to inform how culling should be distributed among management units . We obseThe reduced migration rate from the NC site to KI due to the culling performed on the coast caused the decline of genetic diversity and the increased structuring of mink between these sites. The number of migrants in consecutive years declined (based on Structure assignment). Presumably several factors influence a mink’s ability to traverse the sound and invade the KI. The migrants from the coast were mainly collected on the southern part of the KI archipelago, which is concordant with the prevailing direction of the sea current (from south to north). This suggests that sea current may affect the dispersal potential of mink; individuals from sites on the coast located in the south may have colonized the islands by swimming with the current. This finding also suggests that due to the prevailing surface current direction, from south to north, to further reduce mink colonization of the islands it is advisable to intensify mink culling on the coast located south of KI. The observed pattern exemplifies the usefulness of genetic monitoring to assist in the management of invasive species, by suggesting how to reduce the migration of such species to areas important for biodiversity protection.Interestingly, the recent migration rate was slightly higher from KI to NC than in the opposite direction. This is a rather unexpected result considering that the coast population of mink is probably larger than the population on KI. One possible explanation could be that the food base is limited on the islands after the bird breeding season. A second explanation could be that individuals  disperse from the low density islands to find mating opportunities on the mainland. The majority of migrants recorded in this study were males, which confirms this hypothesis. Moreover, male-biased migration was also observed in another American mink population , and is As expected effective population size was smaller on KI than at both coast sites. The upper 95% confidence interval was between two to six times lower on KI than at the coastal sites. Estimating the effective population size with any precision in large populations , and using a small number of samples or small number of loci is very challenging . HoweverVarious methods of estimating the effective population size may provide different results due to different assumptions , 85. In As the negative impact of non-native invasive species (including American mink) on native biodiversity becomes apparent, there is a growing realization of the need for active management to minimize this impact. Control programmes are increasingly used worldwide, and successful eradication has been achieved mainly on islands (e.g. ), but alS1 Dataset(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 FigThe posterior probabilities averaged across 10 replicate runs at each level of K, proposed clusters (left), and the model value of the second order rate of change of the likelihood function (right). Analysis conducted for: all samples , Koster Islands  and the coast .(PDF)Click here for additional data file.S3 FigBackground map: Europe Base Map—Level 1 Provinces, AND Products B.V. and AND Data Ireland Limited, ESRI.(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 TableFST is given using the adjusted nominal level for multiple comparisons after Bonferroni correction. Sample sizes are given in parentheses. North Coast samples from 2008 and 2009 as well as from 2010 and 2011 were combined.Statistical significance for pairwise (PDF)Click here for additional data file.S2 TableAverage membership higher than 0.4 is shown in grey cells.(PDF)Click here for additional data file.