Vigna umbellate (Thumb.) Ohwi and Ohashi] is an underutilized crop believed to be domesticated in the Myanmar-Thailand region of Asia. In India, rice bean is mainly cultivated in the North-Eastern Hills, which is a hotspot for biological diversity. A 5' anchored PCR was used to develop microsatellite markers in rice bean. Twenty-eight specific primer pairs were designed and employed to characterize sixty five ricebean accessions collected from North East India. A total of 179 alleles were amplified with an average of 6.393 alleles per locus. The gene diversity was high (mean 0.534) in the accessions collected from Darjeeling, Nagaland and Manipur, which are bordering areas with East Nepal and Myanmar, respectively. Exceptionally high outcrossing rate was observed in the entire population. Population structure analysis identified three distinct clusters in which accessions collected from areas bordering Myanmar and East Nepal grouped separately. Using a combination of STRUCTURE and Principal Coordinate Analysis, relative affinity of the intermediate accessions could be established. However, differences in allelic counts among populations were non-significant. The results showed that there is a high level of genetic diversity within the accessions, with high outcrossing rate.Rice bean [ Vigna umbellate (Thunb) Ohwi and Ohashi] is a less utilized grain legume cultivated mainly in Nepal, Bhutan, North East India up to Myanmar, Southern China, Northern Thailand, Laos, Vietnam, Indonesia and East Timor   ,3,4. Thesubgenus , to whicIn India, as a cultigen its distribution is mainly confined to the tribal regions of North Eastern hills and hilly tracts of Western and Eastern Ghats . In the In a study of the genetics of domestication of rice bean, Isemura et al.  reportedSimple sequence repeats (SSRs) or microsatellites are motifs of short tandem repeats that may vary in the repeat units at a given locus. SSRs have specific advantages over other markers as they occur frequently and randomly in all eukaryotic DNA , are mul(AC)n and (GA)n} have been worked out in relatively greater detail  and ii) PCT4 - [5’- KKRVRVR(CT)6−3’]  were used in PCR amplification to isolate a range of repeat motifs. PCR amplification was carried out in a total volume of 20 μl containing 50 ng of template DNA, 2.5 mM MgCl2, 1 X PCR buffer containing 10 mM Tris-HCl (pH = 8.0), 50 mM KCl, 1 unit Taq DNA Polymerase  10 pmol of degenerate primer (PCT2/PCT4) and 0.2 mM of dATP, dGTP, dCTP and dTTP. Amplification was carried out in a thermocycler , with an initial denaturation of 94°C for 3 minutes followed by 35 cycles of 94°C for 30 secs, 58.4°C (PCT2)/ 54.5°C (PCT4) for 40 secs, one minute at 72°C and a final extension step of 72°C for 5minutes. Successful amplification was confirmed by electrophoresing the PCR products on 0.8% agarose gel with a 1kb ladder  as size standard. The PCR products were afterwards purified using a PCR Purification Kit , cloned into pGEM®-T vector  and a library was made. One hundred and twenty four random clones were then sequenced using Bigdye Terminator v3.1 Cycle Sequencing Ready Reaction kit (Thermo Fisher Corporation Inc.) on an ABI 310 genetic analyzer. Microsatellite containing sequences and natures of microsatellites were identified and analyzed using the software MISA [A microsatellite library was constructed using the method of Fisher et al. . BrieflyPrimer modelling was possible for 53 primers pairs, which were then synthesized  and checked for amplification in a set of 12 accessions (2 from each population). Twenty-eight primer pairs that consistently gave unambiguous scorable single bands in all the 12 accessions were later resynthesized with fluorescent labeling  on the f2, 1 X PCR buffer containing 10 mM Tris-HCl (pH = 8.0) and 50 mM KCl, 0.5 unit of Taq DNA Polymerase , two pmol of flourscent dye labeled forward primer, two pmol of unlabeled reverse primer and 0.1 mM of dATP, dGTP, dCTP and dTTP. The PCR profile was: 94o C for 5 minutes followed by 35 cycles of 94o C for one minute, 50.0°C- 60°C (depending on primer) for one minute, 72o C for two minutes, and a final extension step of 72°C for 10minutes. The amplified PCR products were analyzed in an ABI Prism 310 genetic analyzer (Thermo Fisher Corporation Inc.) using GeneScanTM -500 LIZ as size standard.Individual PCR amplifications for each microsatellite were performed using the Genamp PCR 9700 thermocycler (Applied Biosystems.). The PCR amplification was carried out in a total volume of 10 μl containing 10 ng of template genomic DNA, 1.5 mM MgClTM -500 LIZ Size Standard. Autobin v 0.9 software  was used to analyze the raw data. For statistical analysis, the accessions were grouped into six population viz. P1 = Arunachal Pradesh; P2 = Manipur; P3 = Nagaland, P4 = Sikkim; P5 = Darjeeling; P6 = Meghalaya according to their geographic location  of collection. Allelic patterns, analysis of molecular variance (AMOVA), principal co-ordinate analysis (PCoA) and Nei [He) and observed (Ho) heterozygosity, f (fixation index) values were calculated using gDA v1.0 software [t = (1-f)/(1+f)[http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Population structure was analyzed using the software STRUCTURE V.2.3.4 [K = 1–12, and the second run was done at K = 3, K = 6 and K = 8 with 40 replications. Finally, at K = 3, the membership co-efficient from the run with the highest LnP(D) value (-3940.2) was used to assign the accessions to cluster 1–3 (SP1-3) based on their highest membership co-efficient for a cluster. Accessions with <0.6 membership co-efficient for all the clusters were grouped as admixtures (SP4). For the structure clusters (SP1-4), allelic pattern, genetic distance, AMOVA and PCoA were calculated using the GenAlEx V6.5 [Alleles were manually assigned to regular fluorescence peaks, and the sizes of the alleles were calibrated by the GeneMapper Software version 4.0 against GeneScan and Nei  genetic  and Nei . Populatsoftware . Out cro-f)/(1+f). Genetic V.2.3.4 ,37 as de V.2.3.4  except tlEx V6.5 .For the lEx V6.5 .CT (48.98%) and GA (46.9%) were the most abundant type. Repeat units in the dinucleotide varied from 9–25 (both in AG). However, 16 units was the most common (40 in CT and 38 in GA). Among the pentanucleotides, TAAAA motif was the most abundant (14 of 22). A total of 124 clones yielded fragments containing 49 dinucleotides, two trinucleotides and two pentanucleotides. Primer modelling was possible for 53 microsatellite loci out of which 28 microsatellite loci were found to give unambiguous scorable bands. These were selected for further analysis.DNA sequencing of the clones indicated the presence of a total of 124 microsatellite repeats . Di-nuclTm, expected product size and dye labeling used for our study are given in the Details of the developed SSR primers with Ho) was highest in the Manipur population (0.571) and lowest in the Arunachal population (0.495).Mean out crossing was 95.88%.Results of the characterization of the 6 geographic (location of collection) populations with the microsatellite markers are given in Allelic patterns across the six populations are presented in K) with five replicated runs per K value. ‘Structure Harvester’ program (http://taylor0.biology.ucla.edu/) was used to determine the optimum K value based on both LnP(D) and Evanno’sΔK[K = 3 (major peak) and the other at K = 5 . Two minor peakswere also seen atK = 6 and K = 8  values showed that atK = 3, nearly 50% of the accessions could be assigned to one of the three SPs considering a membership coefficient of ≥0.6 for a SP. However, at K = 6 and K = 8, keeping the same parameter, no accession could be assigned to several SPs. Therefore, the run with the highest LnP(D) value at K = 3 was used for assignment of accessions to SPs.Analysis of the population using the model-based approach of the STRUCTURE software indicated the presence of structured population. Initially, the analysis was performed for evaluating 1 to 10 clusters (vanno’sΔK. Two peaat K = 5 . Consideat K = 5 . To overnd K = 8 . Three fΔK method supported the presence of 3 clusters (K = 3), represented here as SP1, SP2 and SP3, respectively. The inferred population structure is given in the The LnP(D) and Evanno’sFst values  of 0.400, 0.441 and 0.650, respectively with an average of 0.497indicating a low to moderate difference among clusters. Net nucleotide distance among SPs was highest between SP1 and SP2 (0.130) and lowest between SP1 and SP3 (0.052). At K = 3, 32 accessions were admixture (<0.6 membership co-efficient for any cluster).The three clusters (SP1-SP3) had population specific Mean allele pattern across the SPs is given in CT/GA) was somewhat similar to the previous reports on genic SSRs (AG/CT) of rice bean [AC/GT was reported to be the most frequent type [In the recent years, a lot of interest has been shown in the assessment of genetic diversity in rice bean and development of molecular markers for the purpose. AFLP , RAPD anice bean  and mungice bean . In the ent type . The proAverage numbers of alleles per locus amplified by the primers reported here are more compared to previously reported SSRs from rice bean  and azuki bean . HoweverThe set of 65 genotypes were grouped into six populations based on their geographic location of collection, and into two regions based on proximity of collection locations. The hills of Arunachal Pradesh, Manipur, Nagaland and Meghalaya are almost adjacent and, therefore, were grouped as a region. The hills of West Bengal (Darjeeling District) and Sikkim share a common border and the collections from there were grouped as another region. The mean gene diversity observed in the present set (0.534) is comparable to the gene diversity (0.535) observed in the cultivated accession of rice bean in a previous study . Howeverviz. MNPL3 used in this study), which are bold seeded. However, rice bean is seldom maintained as a perineal crop. The f (fixation index) values obtained in the 6 geographic location-based populations indicate an exceptionally high out crossing rate, which needs further investigation. Furthermore, the low inter population variation and non-significant differences in allelic counts are probably because of the high outcrossing rate. It is interesting to note that the Manipur population, and the Darjeeling population mostly grouped separately in the PCoA. The Nagaland and Meghalaya populations (Pop3 and Pop6 respectively) showed a tendency to group in these two groups. Half of the Sikkim (bordering Darjeeling) population (Pop 4) also showed grouping with the Darjeeling population. Manipur is an Indian State bordering with Myanmar , repeat motif, annealing temperature and expected product size when run individually. Genebank accession numbers of clones: JQ839228.1 GI: 383479073 to JQ839255.1 GI: 383479100.(DOCX)Click here for additional data file.S1 Fig(a)–Burnin period 5,000, MCMC repeats 50,000, number of replicates 10, clusters 1–10. (b)—Burnin period 10,000, MCMC repeats 100,000, number of replicates 10, clusters 2–6.(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigPop1 –SP1, Pop2 –SP2, Pop–SP3, Pop4 –SP4 (highest membership coefficient for SP1), Pop5 –SP5 (highest membership coefficient for SP2), Pop5 –SP5 (highest membership coefficient for SP3).(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Salaria pavo, a small marine intertidal fish that inhabits rocky habitats of the Mediterranean and the adjacent Atlantic Ocean. We screened 170 individuals using mitochondrial and nuclear sequence data from eight locations. Four models of genetic structure were tested: panmixia, isolation-by-distance, secondary contact and phylogeographic break. Results indicated clear asymmetric migration from the Mediterranean to the Atlantic but only marginally supported the isolation-by-distance model. Additionally, the species displays an imprint of demographic expansion compatible with the last glacial maximum. Although the existence of a refugium in the Mediterranean cannot be discarded, the ancestral lineage most likely originated in the Atlantic, where most of the genetic diversity occurs.Transition zones are of high evolutionary interest because unique patterns of spatial variation are often retained. Here, we investigated the phylogeography of the peacock blenny,  Many terrestrial and marine species have often experienced expanding and contracting range shifts over time . These rDicentrarchus labrax, Diplodus puntazzo and Coryphoblennius galerita have shown high genetic divergence between populations inhabiting both sides of the AOF . The AOF is situated east of the Strait of Gibraltar by the convergent Atlantic and Mediterranean water masses, stretching from Almeria on the Spanish coast to Oran on the Algerian coast. Some species such as  the AOF  while otSalaria pavo, occurs mainly around the Western Mediterranean coasts and from the Bay of Biscay south to the Canaries  and temperature from 1° to 30 °C  panmixia, whereby there is no discernible geographic or otherwise genetic structure corresponding effectively to a random distribution of haplotypes . Sequences for each locus were aligned, edited, and trimmed to a common length using the DNA sequence assembly and analysis software GENEIOUS PRO 7.0 .Total genomic DNA was extracted from fin or muscle samples with the REDExtract-N-Amp kit (Sigma-Aldrich) following the manufacturer’s instructions. Voucher specimens are deposited in ISPA (ethanol preserved tissues). We selected two unlinked genes of different genomes to be sequenced: the mitochondrial D-loop and the nuclear S7 ribosomal protein gene . Nucleafluxus-engineering.com) to determine the genealogical relationships among haplotypes and to consider their geographical distributions. POWSIM 4.1  algorithm , as implWSIM 4.1  was usedFST and ΦST) and one genetic differentiation index (Jost D) because they represent different properties of population partitioning , ϕST/(1 − ϕST) and (D∕(1 − D)). Mantel tests  were performed using mantel.xla 1.2.4  we ran Mantel tests  for pairla 1.2.4 .FCT and ϕCT), the proportion of variation among populations within groups (FSC and ϕSC) and the proportion of variation within populations (FST and ϕST) . A modification of this hypothesis, allowing to consider the existence of other partitions, was also explored with the spatial structure of genetic variation using a Spatial Analysis of Molecular Variance (SAMOVA) (K (number of groups). The SAMOVA was run from K = 2 to the value of K that maximizes the value of the FCT statistic.To test hypothesis 4, the existence of a phylogeographic barrier dividing the Atlantic from the Mediterranean, an analysis of molecular variance (AMOVA) was used to examine the amount of genetic variability partitioned among that barrier . AMOVA c(SAMOVA) . SAMOVA Salaria pavo adults are not known to undertake active migrations, therefore, instead of referring to migration rates (M), we will refer instead to gene flow (G). G and population size parameter (θ) were inferred using the maximum likelihood (ML) in Migrate-n ver. 4.2.6  Computational Cluster Facility  and on the R2C2 research group cluster facility, provided by the IT department of the University of Algarve.r. 4.2.6  among Atximation , as implximation , which wS. pavo was inferred with the Atlantic D-loop data using the coalescent Bayesian skyline plot (BSP) model as implemented in BEAST v. 2.3.1  were obtained. The D-loop data set after alignment consisted of a total of 300 bp comprising 52 polymorphic sites (17%) and 10 (3%) parsimony informative sites. Overall, mtDNA diversity was high, with 49 haplotypes recovered. A large proportion of haplotypes (57%) were singletons, i.e., represented by a single individual. Forty haplotypes (82%) were private, i.e., occurred in only one location. In total, 92% of haplotypes had a frequency lower than five individuals. The most frequent haplotype in the Atlantic was shared by 25 individuals, followed by two haplotypes shared by 13 individuals, one present only in the Mediterranean and the other in both Mediterranean and Atlantic , except Sado which displayed a significantly lower haplotype diversity when compared to Cadiz, Ria Formosa, Olhos d’Água and Barcelona.A total of 131 D-loop sequences  (FST of ≥ 0.0248 (time in generations = 150) could be detected with ≥95% confidence . When FST was set to zero (simulating no divergence among samples), the proportion of α error of type I (rejecting null hypothesis when true) was lower than 5%.The  network  has an o0.00001) . POWSIM FST  and ϕST  but was rejected using D . No haplotype frequency cline (hypothesis 3) could be detected as there were only three haplotypes shared between more than 2 locations.Overall mean pairwise genetic differentiation between the main geographical groups  showed a tendency for higher Atlantic–Mediterranean values . PairwisFCT =  − 0.009, p-value = 0.733; and ϕCT = 0.071, p-value = 0.072), or when we use Barcelona as the sole location representative of the Mediterranean Sea . The SAMOVA results showed that FCT statistic does not increase as the number of groups increased (in FCT were: 2-group (BA)(CG-CA-RF-OA-SA); 3-group (BA)(CG-CA-RF-OA)(SA); 4-group (BA)(CG)(CA-RF-OA)(SA) and finally 5-group (BA)(CG)(CA)(RF-OA)(SA). None of the FCT values were significant, however, the 4-groups arrangement was marginally non-significant .There was no support for hypothesis 4 in  indicatieased in . The locMIGRATE-N was run to determine the level and direction of gene flow across the Almeria-Oran oceanographic boundary. The estimated log Bayes factors based on the Bezier approximation score indicated that the most probable model is the one that contemplates asymmetric migration between the Atlantic and the Mediterranean . The numS. pavo have experienced a long period of demographic stability in the past, followed by a mild decrease of population size and a quick expansion (The Bayesian skyline plot indicated that the Western Mediterranean and Atlantic locations of xpansion . The ploJF834709 –JF834885) were obtained. The S7 nuclear region data set after alignment consisted of 519 characters, with seven polymorphic sites, among which five had ambiguities. Using the ELB algorithm, we defined 12 closely related alleles, with four abundant and almost ubiquitous alleles, and the remainder represented by only one or two individuals. Overall gene and nucleotide diversities were low, 0.69 ± 0.02 and 0.18% ± 0.14%, respectively. The haplotype network  (p > 0.62).A total of 136 S7 first intron sequences (GenBank accession numbers:  network  evidence= 0.618) . Also pa= 0.618)  and non-= 0.618) . The difSalaria pavo (CG)(CA-RF-OA)(SA) arrangement is the one that maximizes FCT, when compared to all other possible groups , and it is also the one that that makes sense from the geographical and isolation-by-distance viewpoint. Barcelona, well in the Mediterranean constitutes a separate group; Cabo de Gata, on the frontier of the Alboran Sea, is also separate; Cadiz, Ria Formosa and Olhão, are all situated in the Atlantic region adjacent to the Strait of Gibraltar, forms another group; and finally, Sado a clearly Northeastern Atlantic location.Panmixia (hypothesis 1) was concomitantly rejected by the haplotype network , the spaulations , we do nDiplodus: D. puntazzo and D. sargus, Salaria pavo displays no significant differentiation across the Atlanto-Mediterranean boundary and this permeability contrasts with the strong across-boundary differentiation displayed by another intertidal blenniid Coryphoblennius galerita  were at most 13 °C in the Iberian Atlantic and most of the west Mediterranean  displays a significantly lower haplotype diversity values than those found in other Atlantic  and Mediterranean locations (Barcelona), which is concordant with a postglacial colonization event.Some fish species display a strong genetic discontinuity between each side of the Almeria-Oran oceanographic front, but this pattern is species-dependent. In the same family  of the S. pavo lineage A (Previously published work hypothesized that the reduced genetic variation detected in ck event . Howeverineage A  suggesteS. pavo in the Atlanto-Mediterranean region is better explained by a combination of some degree of isolation-by-distance and asymmetric migration. The ancestral lineage most probably originated in the Atlantic, where most of the genetic diversity is present. Both dispersal potential and physical factors such as local oceanographic conditions are playing a major role in shaping the genetic structure of this species.In summary, we propose that the genetic pattern of 10.7717/peerj.3195/supp-1Appendix S1Click here for additional data file.


Stichopus chloronotus is an economically and ecologically important sea cucumber species throughout its range. This species is widely distributed, inhabiting coral reefs of the Indo-Pacific Ocean. Our study evaluated population genetic structure and levels of genetic diversity in southern Japan. A total of 180 individuals were collected from eight locations from Okinawa and Okinoerabu Islands and sequenced using mitochondrial 16S ribosomal DNA (16S) and nuclear histone H3 (H3) gene. Only three 16S haplotypes were detected (518 bp) with haplotype diversity ranging from 0 to 0.56 and nucleotide diversity from 0 to 0.1%. H3 showed no variation among the studied locations. It is plausible that such results could be due to a shift to asexual reproduction. Additionally, the presence of the species on the east coast of Okinawa could only be detected in one location and all individuals consisted of a single haplotype. Genetic differences between the east and west coasts of Okinawa have been noticed in other coral reef organisms, and attributed to either ecological or biogeographical historical differences between the coasts due to differing levels of isolation during Pleistocene ice ages. Results from the present study should inform management and conservation policies of S. chloronotus in southern Japan.The greenfish sea cucumber  They are deposit feeders of carbonate sand and rubble through their digestive tracts and dissolve CaCO process . As ‘ecoHowever, sea cucumbers of the order Aspidochirotida (families Holothuroidea and Stichopodidae) are suffering from overexploitation and overfishing in several countries  due to hStichopus chloronotus Brandt, 1835 (greenfish sea cucumber), belonging to the family Stichopodidae, is a member of such highly commercial marine sea cucumber fisheries. This species is a widely distributed across most of the tropical Indo-Pacific region, from the Western Indian Ocean to the Central Pacific, and from southern Japan to northern Australia  and asexually by transverse fission  using sequences of mitochondrial DNA  and nuclear DNA (histone H3), in order to provide genetic connectivity. We examine whether S. chloronotus on the east and west coasts of Okinawa are genetically differentiated as has been observed in a number of recent studies on other sea cucumber  and one location at Okinoerabu Island, Kagoshima, Japan . Samples of S. chloronotus were obtained non-lethally through body wall-tissue biopsy (1 cm2) as in our previous study , in both forward and reverse directions.Total genomic DNA was extracted from a small piece (0.25 g) of body wall tissue using a DNeasy Blood & Tissue extraction kit  following the manufacturer’s protocol. Polymerase chain reactions (PCR) were used to amplify fragments of two DNA markers; mitochondrial 16S and nuclear H3 gene, using the following primers: 16SA-R 5′-CGC CTG TTT ATC AAA AAC AT-3′; 16SB-R 5′-GCC GGT CTG AAC TCA GAT CAC GT-3′ , and H3ahttp://www.geneious.com, h), haplotype diversity (hd) and nucleotide diversity (π) were estimated with DNASP v.5.10.01  for all pairs of locations using Arlequin 3.5.1.2  from eight total locations around Okinawa and Okinoerabu Islands (hd = 0.36), and was highest in the Oyama (hd = 0.558) and Ryugu (hd = 0.5143) locations. In addition, the nucleotide diversity of 16S was low for all locations combined   was rejected, results of the hierarchical AMOVAs did not support significant differences between east and west coasts or between north (Okinoerabu Island), east of Okinawa Island (Teniya) and west of Okinawa Island  (Significant pairwise Φ < 0.01) . Most of, Zanpa) .ST for 16S revealed three clusters; (1) Yona, Zanpa, Motobu, Itoman and Okinoerabu, (2) Ryugu and Oyama, and (3) Teniya  using overall pairwise Φ) Teniya . S. chloronatus impose some caution in the overall interpretation of results. Various studies of S. chloronotus across regions using different genetic markers have also revealed low genetic diversity.  S. chloronotus in some locations such as the Great Barrier Reef, Torres Strait, and La Reunion  and nuclear H3 genes  and small individual sizes (caused by over-exploitation) have been identified as the most important factors that can lead to asexual reproduction in sea cucumbers . Over thxploited . SimilarS. chloronatus individuals on the east coast, however, Teniya was the only location on this coast where the species could be found, further indicating the possibility of the above mentioned factors. Furthermore, our results indicated the presence of gene exchange among all Okinawa Island west coast locations and Okinoerabu Island. This pattern is consistent with the route of the Kuroshio Current flowing northward to the west of Okinawa Island  in southern Japan are needed to confirm this assertion. Similar patterns of genetic differentiation between east and west Okinawa Island locations have been observed in recent studies on the sea cucumber Holothuria edulis (Leucothoe vulgaris (Acropora digitifera), Okinawa Island locations from both east and west coasts showed no separation and were treated as one panmictic population (A. digitifera (approx. 45 days; The haplotype frequency differences between the west coast of Okinawa and the single location on the east coast may be due to the existence of habitat differences, oceanographic barriers to dispersal promoting a reduction of larval exchange, and/or historical factors. We extensively searched for a Island , and suga edulis  and in tvulgaris . White, vulgaris  and/or gvulgaris . In contpulation . Differepulation  than in S. chloronotus indicates low levels of genetic diversity and reduced genetic exchange among sites in Okinawa and Okinoerabu islands, and particularly between the east and west coasts of Okinawa Island. These results will be useful for fisheries management decisions regarding this species in southern Japan. Both In conclusion, the present genetic survey of


Anopheles gambiae (s.s.) is crucial to inform genetic control and manage insecticide resistance. Unfortunately, species characteristics such as high nucleotide diversity, large effective population size, recent range expansion, and high dispersal ability complicate the inference of genetic structure across its range in sub-Saharan Africa. The ocean, along with the Great Rift Valley, is one of the few recognized barriers to gene flow in this species, but the effect of inland lakes, which could be useful sites for initial testing of genetic control strategies, is relatively understudied. Here we examine Lake Victoria as a barrier between the Ugandan mainland and the Ssese Islands, which lie up to 60 km offshore. We use mitochondrial DNA (mtDNA) from populations sampled in 2002, 2012 and 2015, and perform Bayesian cluster analysis on mtDNA combined with microsatellite data previously generated from the same 2002 mosquito DNA samples.Understanding population genetic structure in the malaria vector Hierarchical analysis of molecular variance and Bayesian clustering support significant differentiation between the mainland and lacustrine islands. In an mtDNA haplotype network constructed from this and previous data, haplotypes are shared even between localities separated by the Rift Valley, a result that more likely reflects retention of shared ancestral polymorphism than contemporary gene flow.An. gambiae on the Ssese Islands, their small size, level terrain and ease of access from the mainland, the relative simplicity of the vectorial system, and the prevalence of malaria, are all attributes that recommend these islands as possible sites for the testing of genetic control strategies.The relative genetic isolation of  Anopheles gambiae (s.s.) (formerly An. gambiae S-form) is a principal vector of malaria in sub-Saharan Africa, where 91% of an estimated 445,000 malaria deaths worldwide occurred in 2016 , relatively small size and flat terrain of the islands, and logistical ease of access from the mainland, may all be considered advantages in the initial testing phases. Unfortunately, even by Ugandan standards, there is also a heavy burden of malaria on these islands. Compared to urban areas of Uganda, where malaria prevalence in children 6–59 months is 11.5%, malaria prevalence in rural areas is considerably higher, reaching nearly 35% on the mainland and 44% in the islands of Lake Victoria [An. gambiae mosquitoes.The most recent assessment of malaria control in Africa using currently available tools is not all good news . Hard-woVictoria . As such


Ambrosia artemisiifolia L.) is an invasive, wind‐pollinated plant nearly ubiquitous in disturbed sites in its eastern North American native range and present across growing portions of Europe, Africa, Asia, and Australia. Phenotypic divergence between European and native‐range populations has been described as rapid evolution. However, a recent study demonstrated major human‐mediated shifts in ragweed genetic structure before introduction to Europe and suggested that native‐range genetic structure and local adaptation might fully explain accelerated growth and other invasive characteristics of introduced populations. Genomic differentiation that potentially influenced this structure has not yet been investigated, and it remains unclear whether substantial admixture during historical disturbance of the native range contributed to the development of invasiveness in introduced European ragweed populations. To investigate fine‐scale population genetic structure across the species' native range, we characterized diallelic SNP loci via a reduced‐representation genotyping‐by‐sequencing (GBS) approach. We corroborate phylogeographic domains previously discovered using traditional sequencing methods, while demonstrating increased power to resolve weak genetic structure in this highly admixed plant species. By identifying exome polymorphisms underlying genetic differentiation, we suggest that geographic differentiation of this important invasive species has occurred more often within pathways that regulate growth and response to defense and stress, which may be associated with survival in North America's diverse climatic regions.Common ragweed ( Ecosystem disturbance by human activity has substantially altered the evolutionary trajectories of many plant species. The evolutionary mechanisms that grant some plants the ability to become invasive arise from complex, synergistic processes. These include rapid adaptation to novel environments  has experienced data remarkable success in Europe, providing opportunities to study the population genetic differentiation between native and introduced ranges  and estimation of genetic clustering with genotyping‐by‐sequencing (GBS) data, we explore the genomic basis of geographic differentiation in native‐range common ragweed populations. Our approach thus seeks to evaluate the contemporary population structure inferred by Martin et al. , by estiA. artemisiifolia individuals from 37 locations were sampled in 2009‐2010, predominantly from roadsides and agricultural fields across the eastern portion of the species' native range (Table S1). Leaf tissue samples were immediately desiccated by collection into silica gel. Silica‐dried leaf tissue was pulverized with silicon beads in a Qiagen  TissueLyser® and then extracted using plant‐specified reagents with the AutoGen  AutoGenprep 965 Automated DNA Isolation System.A. artemisiifolia genomic DNA extracts were digested with the restriction enzyme ApeKI (4.5‐bp recognition site 5′‐GCWGC‐3′). Following ligation of unique, individual‐specific indexing adapters, libraries were pooled along with other samples from a different study and multiplex sequenced on two flowcell lanes (95 samples and one blank per lane) on the Illumina  HiSeq2000 platform using 100‐bp Single Read chemistry.Genotyping‐by‐sequencing (GBS) library construction was performed by the Institute for Genomic Diversity and Computational Biology Service Unit at Cornell University  according to a published protocol  employing network analysis and filtration . Consequently, we further filtered the data set of genotypes by removing all individuals for which fewer than 10,000 SNP positions had been genotyped, leaving 60 individuals from 23 sampling locations. We then filtered loci by removing all loci genotyped in at least 50% of the remaining individuals. These filtering steps yielded 6337 SNP loci, genotyped in 60 individuals, and with a per‐individual mean read depth of 10.8X (Table S1). This 6337‐SNP data set was used in later enrichment tests.r2 ≥ 0.5). However, a majority of SNPs were in strong LD with at least one of the other 6336 SNPs. Thus PLINK‐facilitated LD pruning removed a large portion of the data, and subsequent analyses of genetic structure were performed on a multiple sequence alignment of 2829 SNP sites assessed in 60 individuals spanning eastern North America.Because the presence of loci in genetic linkage in a genomic data set can lead to the detection of spurious genetic structure, we used PLINK v1.09  using SMARTPCA within the software package EIGENSOFT v4.2 . For each value of K, the replicate of highest likelihood was chosen for further analysis as well as plotting.ADMIXTURE v1.22  was assessed via permutation tests (n = 99) in which individual cluster assignments were randomly permuted. The R package pegas v0.8.1  flanking sequenced ApeKI restriction sites associated with these SNPs were used in a BLASTALL v2.2.26  protein database with an E‐value threshold of 10Ambrosia species are known for an abundance of diversity of terpenoid compounds in their tissues , which is consistent with both nonequimolar sample pooling and unequal library performance in the sequencing run. We note that A. artemisiifolia genome (1.1 Gbp/(44.5) = 2.15M; Kubešová et al. Sequence data processing with the UNEAK pipeline revealed that 205.5M  reads carried both the ApeKI restriction site and an unambiguous barcode. The number of unique diallelic, single‐polymorphism tags identified by TASSEL's network analysis was 4,824,895, which is approximately equal to twice the expected number of ApeKI cut sites in the ~1.1‐Gbp A. artemisiifolia populations and have been discussed extensively  was low and half that of the western and southeastern clusters .To reduce the dimensionality of our genomic SNP data set and to obtain initial view of genetic structuring in our data, the 2829 filtered, LD‐pruned SNP variants from 60 individuals were used to perform a PCA. This analysis revealed that the first two principal component axes reflect the geography of wild populations remarkably well, while accounting for around 6% of the samples' genomic variance Fig. . IndividK approaching the number of individuals in the data set . Implementation of the Evanno et al. , although the assigned proportions of genetic ancestry were strongly correlated with geography for K = 2–5  whose diffuse boundary is approximately aligned along the axis of the Appalachian Mountains. The ancestries of individuals from Arkansas, Louisiana, and Tennessee indicate roughly 50% admixture.Estimation of ancestry proportions with ADMIXTURE showed that a single, panmictic genetic cluster is the most likely, although lower cross‐validation errors were achieved with very high values of o et al.  method r–5 Figs. . When K K = 3, the eastern cluster subdivides into northeastern and southeastern clusters, and the three genetic clusters are more highly differentiated (pairwise FST = 0.012–0.019) than the K = 2 clusters (Table S3). The southeastern cluster is the most divergent and encompasses individuals from Florida, Louisiana, Georgia, and Arkansas, with mixed ancestry appearing in all populations except those in Florida. When K = 4, the western genetic cluster subdivides into southwestern and midwestern clusters that are mixed throughout the western range and more poorly correlated with geography. These four clusters are the most highly differentiated , with the midwestern cluster most divergent (Table S4). At K = 5, individuals collected in Louisiana are assigned their own ancestral cluster, and at K > 5, geographic correlations appear to break down.When The 5% top‐weight loci from the principal components analysis were represented by 632 tag sequences, 38–64 bp in length. The BLAST analysis aligning these sequences with the native transcriptome assembly identified 222 transcript sequences with unambiguous homology to the query tag sequences. Transcripts from this data set were also enriched for particular GO terms of varied function, including defense response, biosynthesis and metabolism of triterpenoids, catabolysis of glucosinolates (aromatic compounds related to defense against pathogens and herbivores), regulation of growth, RNA metabolism, sulfur/phosphorus metabolism, response to the plant growth hormone indolebutyric acid, processes relating to protein localization to the mitochondrion, and drug transport/responses Table .A. artemisiifolia experienced rapid evolution upon introduction to Europe, increasing its success as an invasive after multiple introductions from different source populations , Atlantic white cedar  and biosynthesis of plant secondary compounds (defense against pathogens and herbivores) were previously identified as major factors in gene expression differences between introduced ragweed populations and native populations sampled from southeastern Canada and midwestern USA  error for different numbers of assumed ancestral population clusters (K).Figure S7. Implementing the Evanno et al. (K genetic clusters.o et al.  method fTable S1. Provenance of A. artemisiifolia individuals included in this study.Table S2. Pairwise FST estimated between three genetic clusters defined by principal components analysis.Table S3. Pairwise FST estimated between genetic clusters defined by ADMIXTURE analysis with K = 3.Table S4. Pairwise FST estimated between genetic clusters defined by ADMIXTURE analysis with K = 4.Click here for additional data file.


Sterechinus neumayeri. We studied the patterns of genetic diversity and structure in this broadcast-spawner across three Antarctic regions: Antarctic Peninsula, the Weddell Sea and Adélie Land in East Antarctica. Genetic analyses based on mitochondrial and nuclear markers suggested that S. neumayeri is a single genetic unit around the Antarctic continent. The species is characterized by low levels of genetic diversity and exhibits a typical star-like haplotype genealogy that supports the hypothesis of a single in situ refugium. Based on two mutation rates standardized for this genus, the Bayesian Skyline plot analyses detected a rapid demographic expansion after the Last Glacial Maximum. We propose a scenario of rapid postglacial expansion and recolonization of Antarctic shallow areas from a less ice-impacted refugium where the species survived the LGM. Considering the patterns of genetic diversity and structure recorded in the species, this refugium was probably located in East Antarctica.One of the most relevant characteristics of the extant Southern Ocean fauna is its resiliency to survive glacial processes of the Quaternary. These climatic events produced catastrophic habitat reductions and forced some marine benthic species to move, adapt or go extinct. The marine benthic species inhabiting the Antarctic upper continental shelf faced the Quaternary glaciations with different strategies that drastically modified population sizes and thus affected the amount and distribution of intraspecific genetic variation. Here we present new genetic information for the most conspicuous regular sea urchin of the Antarctic continental shelf,  The marine benthic fauna of the Southern Ocean is unique and considered to be the most isolated on the planet ,2,3,4,5.The gradual onset of colder conditions with subsequent large-scale glaciation in Antarctica since the early Oligocene has been pivotal to shape the evolution of the Antarctic biota ,15,5,16.At least 38 glaciation cycles have been identified over the last 5 million years by sediment core data ,23. The In situ persistence in Antarctic glacial refugia; (ii) Survival in peri-Antarctic islands or in sub-Antarctic areas; (iii) Retreat to the shelf slope and deep-sea habitats. The in situ persistence scenario suggests the presence of one or several isolated refugia on the shelf associated with strong population bottlenecks. Under this scenario, a species should be characterized by low levels of genetic diversity and by a star-like haplotype network with short genealogies in the case of a single refugium [Trematomus bernacchii and Trematomus pennelli [Nacella concinna [Colossendeis megalonyx [Doris kerguelensis [Nymphon australe [Promachocrinus kerguelensis [However, the response of species to climate change does not necessarily depend on life-history traits, but also on the availability of suitable habitats, that may not allow the species to survive. For marine species living on the Antarctic continental shelf, glacial periods would have corresponded to maximum contraction in geographical range ,43. The refugium ,47. Morerefugium . A scenarefugium . It assurefugium ,46,49. Ipennelli , and theconcinna  and for egalonyx . The retegalonyx ,52, and uelensis  and the australe ,53. In suelensis .Sterechinus neumayeri . This species is found from the shallow subtidal zone down to 500 m depth, but is mainly distributed in the inner and mid continental shelf [S. neumayeri individuals were identified as S. agassizii, a congeneric species with a broad distribution in the Antarctic and Subantarctic . Sterechinus neumayeri is a broadcast-spawner with free-living larvae that settles in the water column after 4 months [S. neumayeri represents a suitable model for studies in reproductive biology [S. neumayeri on the Antarctic continental shelf has been associated with depth, sea ice coverage and sea surface temperature [One of the most conspicuous and endemic inhabitants of the Antarctic continental shelf is the regular sea urchin species al shelf ,56. It hal shelf . However4 months . Due to  biology , embryol biology ,60,61, e biology ,64,65,66 biology ,68 and t biology . Howeverperature , but theS. neumayeri from the Western Antarctic Peninsula (WAP) and East Antarctica (Adélie Land) that could reflect the existence of past or present connectivity sufficient to impede any divergence processes around the continent. Considering that the extension of ice sheets during the LGM may have dramatically affected S. neumayeri populations in shallow waters, the lack of phylogeographic structure could rather be a consequence of a postglacial demographic expansion scenario following a bottleneck or even to a founder effect related to postglacial recolonization. In this study, we analyzed the effect of the recent climatic history of the Southern Ocean on the evolutionary history of S. neumayeri through genetic structure and demographic inference analyses, using both mtDNA and microsatellites markers.Molecular approaches are helping us to understand patterns of genetic diversity and structure better and to infer historical and contemporary demographic processes. Díaz et al.  describeSterechinus neumayeri as study model, a common sea urchin species from the continental shelf of the Antarctic Continent. The species is considered as an Antarctic marine living resource; therefore it is protected by the Commission for the Conservation of Antarctic Marine Living Resources CCAMLR. Permission to collect specimens was issued by the Chilean Antarctic Institute (INACH). Instituto de Ecología y Biodiversidad (IEB/15-2015) ethics committee, which approved sampling protocols and experiments. We complied with local legislation and the Convention on Biological Diversity.This study was conducted using S. neumayeri were deposited in GenBank under the accession numbers MG783406-MG783567.Individuals were collected between 2004 and 2012 in shallow waters at four localities across two major regions including West Antarctica: a) Antarctic Peninsula (AP): James Ross Island, East AP (JRI 64°18´53"S—57°07´53" W: n = 8); Paradise Bay, West AP (PB 64°51´05"S—62°54´36" W: n = 10); Rothera Station, Adelaide Island, West AP (RO 67°42´55”S—68°04´30” W: n = 19); and b) the Weddell Sea  was amplified with specific primers following PCR conditions described by Díaz et al. . PCR ampS) for each region and for the whole dataset to measure whether data deviate from expectations under a mutation-drift model with DnaSP version 5.00.07 [S. neumayeri, a clustering method was used to estimate the number and the composition of panmictic groups, as well as the spatial boundaries between them using a Bayesian model computed with the GENELAND package, version 4.0.0 [6 MCMC iterations were sampled each 1000 steps with a 500 step burn-in period, and a maximum number of clusters K = 10 were run to estimate the model parameters and posterior probabilities of group membership.Levels of genetic polymorphism were determined using standard diversity indices: number of haplotypes (k), number of segregating sites (S), haplotype diversity (H), average number of pairwise differences (Π) and nucleotide diversity (ᴨ) for each locality, for each region and for the whole COI sequence set using DnaSP, version 5.00.07 . We perf 5.00.07 . To have 5.00.07 . Extrapoon 4.0.0  in the Ron 4.0.0 ). This sS. neumayeri were characterized using median-joining networks computed with the program Network 5.0.0.1. [-1. Substitution rates were modified to a tenfold evolutionary rate (10% per million years: 5.1 and 7.2%), considering the correction for time dependence of molecular rates at the population level proposed by Ho et al. [8 generations (sampled every 1000 iterations), discarding the first 10% of the trees as burn-in. The convergence of runs was confirmed with Tracer v1.0.1 [The genealogical relationships among COI haplotypes for each region and for the whole dataset of 5.0.0.1. . Past po5.0.0.1. . In addi5.0.0.1. . We cond5.0.0.1. ), previo5.0.0.1. , for two5.0.0.1. : 0.51 ano et al. . It is io et al. ,89. Geneo et al. ,90,91. Eo et al. ,93, it ho et al. , Antarcto et al.  and Gento et al. . The twor v1.0.1 , ensurinMicrosatellite loci were developed by ATG GENETIC INC. using genomic enrichment and plasmid cloning methodologies . BrieflyPolymerase chain reaction (PCR) amplification mixtures (15 μl) contained 10 ng template DNA, 5 pmol each primer, 250 μM dNTPs, 1.5 μL 10 X PCR buffer and 0.5 U Taq DNA polymerase (Invitrogen). Cycling conditions consisted of an initial denaturing step of 5 min at 94°C, followed by 35 cycles of 1 min at 94 °C, 30 s at the specific annealing temperature , 1 min aBecause it was not possible to amplify the Weddell Sea samples at any loci, we evaluated the genetic differentiation between Antarctic Peninsula localities (West Antarctica) and Adélie Land (East Antarctica) for a total of 73 individuals. This reduced number of individuals was due to difficulties in the amplification of each locus (probably associated with the presence of degraded DNA).2, an estimator of the two-locus heterozygosity disequilibrium over all pairs of loci, under the assumption of inbreeding and linkage equilibrium. P-values for the null hypothesis s = ĝ2 = 0 were obtained by resampling single-locus heterozygosity among individuals within the regions 1000 times (α = 0.05).Genetic diversity analyses were conducted on 73 individuals. The presence of null alleles and scoring errors was checked with MICROCHECKER v.2.2.3 . All nonST) was estimated in GENETIX for each locus separately and overall, and tested for significant differentiation from zero by a permutation test also implemented in GENETIX. These estimates and tests were conducted 1) considering the whole dataset  and 2) after removing individuals with missing data for two loci . STRUCTURE v2.3.3 [S. neumayeri around Antarctica. Two different runs were done with K from 1 to 5; a first run with the whole dataset and a second run with 65 individuals , in order to reduce the possible error that can be inferred from the amount of missing data. Eleven independent replicates with one million Markov chain Monte Carlo (MCMC) analyses were performed for each run with a 100,000 burn-in period under the “admixture model”.Two independent analyses were performed for the structure analyses between the two major biogeographic areas of the Antarctic continent. The fixation index . No insertion/deletion or stop codons were detected in the sequence set. Sequences were not saturated at any position and two amino acid changes were recorded  compared to the mean G-C content (42.4%). Sterechinus neumayeri exhibited low levels of genetic diversity across the Antarctic continent, even though at first glance Fildes Bay in Antarctic Peninsula exhibited higher levels of diversity measured as the number of polymorphic sites (S) and the number of haplotypes (k). Rarefaction analysis showed similar values of haplotype number among localities of Antarctic Peninsula except in James Ross Island and Paradise Bay  and, Adélie Land showed a higher haplotype number than the rest localities (and regions). The Weddell Sea population showed only two polymorphic sites and three haplotypes while Adélie Land in east Antarctica exhibited higher levels of genetic diversity. Haplotype diversity (H), average number of nucleotide differences (Π) and mean nucleotide diversity (ᴨ) were low in the three regions but the lowest values were detected in the Antarctic Peninsula. Despite being the region with the lowest number of samples analyzed, the Weddell Sea showed the highest levels of haplotype diversity. Similarly, Adélie Land showed the highest levels of average number of nucleotide differences, as well as nucleotide diversity . At posiversity .The model based on the Bayesian clustering algorithm implemented in Geneland detected a single cluster for the data set (K = 1), with high values of posterior probability of cluster membership (c.a. P = 0.7), suggesting that there is no genetic structure for the species around Antarctica.S. neumayeri included 23 different haplotypes and showed a typical star-like topology with a very short genealogy: a central haplotype occurring in all regions in high frequency (H1 = 85.1%), from which many low frequency haplotypes radiate separated by one (for the majority) to 2 substitutions. A total of 21 singletons were recorded in the species , the median-joining haplotype network of  species . As expeS neutrality tests were negative for the three regions as expected for star-like topologies, but were significant only for AP and AL  and a second haplotype of low frequency recorded at in the Antarctic Peninsula and the Weddell Sea H10, .The AdélP and AL . SimilarP and AL .S. neumayeri across the Antarctic continent. The time for the most recent common ancestor (trmca) appears between 6–8.4 ky while the onset of the population expansions occurred between 4.7–6.6 ky  was significant (p < 0.001), revealing a strong deviation from HWE. Significant departure from HWE was detected in three of the five loci along the Antarctic Peninsula (P1St–P2St–N4St) while two loci were significant for the east Antarctica AL (P1St–P2St), revealing heterozygote deficiency  = 0.235, p> 0.05 and; AP: ĝ2 = -0.0008, s(ĝ2) = 0, p> 0.05). Thus the Wahlund effect, the presence of two or more genetically differentiated populations in the sample arises as a possible cause, but the missing data may also have been responsible. Moreover, Fildes Bay has an absence of private alleles . Overall heterozygote deficiency  was consistent with pattern observed using microsatellites. Similar patterns of genetic homogeneity over a broad geographical range have been reported in other groups of Antarctic invertebrates including Parbolasia corrugatus [Chorismus antarcticus [Nacella concinna [Parbolasia corrugatus and Chorismus antarcticus shared haplotypes among the Antarctic Peninsula, Ross Sea and Weddell Sea [Chorismus antarcticus, Nacella concinna and Nematocarcinus lanceopes [S. neumayeri lies in the prolonged free-living larval period recorded in the species, which allows transport over large distances [S. neumayeri larvae described by Marsh et al. [S. neumayeri [S. neumayeri may have been facilitated principally by the current near-shore areas ACoC.As previously proposed by Díaz et al.  for S. nrrugatus , Chorismarcticus  and Naceconcinna ,33,49. Mdell Sea ,104. Thedell Sea . Haye etdell Sea  recognizanceopes . The disistances . Gene flh et al. , could aeumayeri . Díaz eteumayeri  proposedeumayeri ), which eumayeri ) which sS. neumayeri suggests that this species was strongly impacted by the recent Antarctic Quaternary climate history. Similar genetic diversity patterns have been reported in other Antarctic echinoderms including the irregular sea urchin Abatus agassizii [Ophionotus victoriae [Odontaster validus [Promachocrinus kerguelensis [Parbolasia corrugatus [Chorismus antarcticus [Eusirus [Nacella concinna [Nymphon australe [Pareledone turqueti [The remarkable characteristics of the extant shallow benthic fauna of the Southern Ocean fauna are its diversity and long-term persistence despite to the Quaternary glacial cycles ,43,110. gassizii , the briictoriae , the sea validus  and the uelensis , as wellrrugatus , the malarcticus , amphipo[Eusirus , the Antconcinna , the seaaustrale  and the turqueti .S. neumayeri may have survived in the deeper areas during the LGM is difficult to demonstrate or to test. However, the presence of the congeneric S. antarcticus on the outer Antarctic continental shelf (below 300 meters depth), and the restricted area in which both species do overlap [S. antarcticus in deeper water may have impeded a habitat switch of S. neumayeri to less ice-impacted deeper areas during the LGM. Consequently, and together with the pattern of genetic diversity and structure detected in this study, the most plausible hypothesis explaining the resiliency of S. neumayeri in the Antarctic would be the in situ shelf refugia. Evidence for survival in situ during the glacial periods are common for the Northern Hemisphere, where some small ice-free refugia have been identified through the study of genetic diversity and structure. Maggs et al. [S. neumayeri characterized by a single dominant haplotype around Antarctica together with numerous rare haplotypes differing by one or two substitutions is congruent with the pattern of genetic diversity proposed by Allcock & Strugnell [Parbolasia corrugatus [Chorismus antarcticus [Nacella concinna survived in South Georgia Island [The possibility that a species with shallow distribution such as  overlap  may suggs et al.  distingus et al. ). The ‘strugnell  for spectrugnell ), while trugnell  has alsorrugatus  and the arcticus  that woua Island .S. neumayeri is congruent with this hypothesis, but in the Antarctic region, Adelie Land (East Antarctica) displayed the highest nucleotide diversity (0.00115) and suggests this area as a potential refuge for the species. This area has also been proposed as glacial refugium for the crinoid with a planktonic stage Promachocrinus kerguelensis [Eusirus [Nymphon australe [Pareledone turqueti [Ice-free regions existed in a range of temporal and spatial scales and are potential refuges during glacial cycles. Evidence for the diachrony of ice-sheet extensions around Antarctica resulting in the incomplete coverage of shelf areas by grounded ice  have beeuelensis , amphiposity 0.00 and suggaustrale  and the turqueti . Parts oturqueti . The preturqueti  due to tturqueti .S. neumayeri, mainly through the main oceanographic currents that allow long distance dispersal of pelagic larvae around the Southern Ocean [Nacella concinna [Nematocarcinus lanceopes [Nymphon austral [Limacina antarctica [During the Early-Middle Holocene , the degrn Ocean ,130,131.rn Ocean ,36,132 aconcinna , Nematocanceopes , Nymphon austral  and Limatarctica .S. neumayeri in the Southern Ocean and some unique characteristics like the endemism and extensive distribution along the Antarctic shelf. Our results suggest a circum-Antarctic connectivity strong enough to impede any divergence processes around the continent. Under a postglacial scenario of recent demographic expansion, our results also suggested that the extension of ice sheets during the LGM may have drastically reduced the habitat and population sizes of S. neumayeri. Because S. neumayeri is endemic to the upper Antarctic continental shelf, the star-like topology with a central dominant haplotype, low genetic diversity and recent demographic expansion signal supports the hypothesis of a single in-situ refugium. We propose a scenario of rapid postglacial recolonization of shallow Antarctic areas from one area less ice-impacted where it survived through the LGM, probably in the eastern part of the continent.Climatic and oceanographic processes together with life history traits are major factors explaining the evolutionary history of S1 Table(DOCX)Click here for additional data file.S1 FigA) Boxplot for the assignment of individual genotypes for K = 1 to K = 5; B) Graph that shows the variation of the mean Ln probability of data.(TIF)Click here for additional data file.


The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1), a non-receptor protein tyrosine phosphatase, has been reported as a negative regulator of phosphorylated signal transducer and activator of transcription 3 (STAT3) and linked to tumor development. In this present review, we will discuss the importance and function of SHP-1/p-STAT3 signaling in nonmalignant conditions as well as malignancies, its cross-talk with other pathways, the current clinical development and the potential role of inhibitors of this pathway in anticancer therapy and clinical relevance of SHP-1/p-STAT3 in cancers. Lastly, we will summarize and highlight work involving novel drugs/compounds targeting SHP-1/p-STAT3 signaling and combined strategies that were/are discovered in our and our colleagues’ laboratories. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor which functions mainly through dimerization upon phosphorylation at tyrosine residues and translocation to cell nuclei ,2. STAT3Oncogenic STAT3 hyper-activation has been observed in various malignant human tumors, including lung, breast, colon, liver, prostate, stomach, pancreas, kidney and brain cancers ,11,24,25SHP-1 gene silencing by forming complexes with DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) at the promoter region of SHP-1 [SHP-1 gene silencing [To prevent inappropriately sustained STAT3 activation, there are multiple intrinsic regulators that can timely suppress STAT3 pathway. SHP-1 is one of PTP members that can accomplish this important function . SHP-1 hof SHP-1 . Noteworilencing . Taken tThe central link of STAT3 with other key oncogenic pathways such as activated protein 1 (AP-1) signaling complex, nuclear factor NF-κB and Wnt/β-catenin signaling makes STAT3 an attractive target and a master regulator for a plethora of cellular functions. For example, STAT3 regulates a broad range of transcription factors, such as c-fos, whereas c-fos is a key member of AP-1 proteins, a key cell life and death regulator . The comArtemisia capillaris induces SHP-1 to downregulate the expression of Jak1/2 and STAT3, and STAT3-regulated genes that mediate cell proliferation, cell survival, invasion, and angiogenesis [SHP-1-mediated STAT3 downregulation is an appealing anti-cancer strategy to induce apoptosis in cancer cells. Previously, sorafenib has been demonstrated that can induce apoptosis in cancer cells through a novel kinase inhibition-independent mechanism. Sorafenib is the first  and is sogenesis .Apart from the above-mentioned drugs, there is a range of other anticancer compounds like Icariside II , betulinDespite these promising results, SHP-1 agonist in combination with current approved chemotherapeutic agents may be synergistic to overcome toxicity, drug resistance or other side effects associated with high doses of single drugs. Sorafenib was the first and remains the only approved targeted therapy for advanced HCC in 2016. Comparison with single-agent sorafenib, sorafenib in combination with SC-43 had a synergistic effect on the increment of SHP-1 activity and the decrement of p-STAT3 in HCC cells . In addiNotwithstanding, the prognostic role of p-STAT3 on cancer patient outcome seems to be conflicting among various solid cancers . Thomas In contrast to the relatively established regulatory roles of SHP-1 in immune and hematopoietic cells ,114, theCurrently, SC-compounds (SHP-1 agonists) are still in the pre-clinical stage, and none of these compounds have yet been approved as investigational new drugs (IND) for cancers. In contrast, STAT3 inhibitors that are designated based on more common strategies for inhibiting STAT3 transcriptional activity such as JAK inhibitors, STAT3 dimerization domain inhibitors, STAT3-DNA binding blockers, and antisense oligonucleotide inhibitor of STAT3 have been in various developmental stages or in clinical trials for cancer treatment (reviewed nicely in references ,9,10,109Loss of SHP-1 contributes to the activation of JAK/STAT3 as well as the other oncogenic pathways, and may further spark off an oncogenic feedforward loop to amplify tumorigenic signals. Hence, reactivating the function of SHP-1 phosphatase to target STAT3 can be a promising candidate for targeted cancer therapy and lay the foundation of new drug discovery. It is worth mentioning that SHP-1 has been reported as a potential target for cancer immunotherapy ,121. LosThe potential combination strategies of SHP-1 agonists and current approved chemotherapeutic drugs may also provide further insights into mechanisms of synergy and/or resistance, possibly leading to the development of clinical trials in human cancers. Analogously, abnormal SHP-1/p-STAT3 signaling can be a useful prognostic biomarker for predicting chemotherapeutic drugs’ response. These findings demonstrated an improved understanding of SHP-1/p-STAT3 interaction in promoting cancer progression and these concepts can be further translated into efficacious combination approaches to prolong the survival rate and improve the quality of life in patients. Future studies may help to delineate the impact of targeting SHP-1/STAT3 on the network among these pathways.


Vulpes lagopus) is a circumpolar species inhabiting all accessible Arctic tundra habitats. The species forms a panmictic population over areas connected by sea ice, but recently, kin clustering and population differentiation were detected even in regions where sea ice was present. The purpose of this study was to examine the genetic structure of a population in the High Arctic using a robust panel of highly polymorphic microsatellites.The arctic fox  revealed the presence of genetic subdivisions. Overall, the sPCA revealed two spatially distinct genetic clusters corresponding to the northern and southern parts of the study area, plus another subdivision within each of these two clusters. The north–south genetic differentiation partly matched the distribution of a snow goose colony, which could reflect a preference for settling into familiar ecological environments. Secondary clusters may result from higher-order social structures (neighbourhoods) that use landscape features to delimit their borders. The cryptic genetic subdivisions found in our population may highlight ecological processes deserving further investigations in arctic foxes at larger, regional spatial scales.The online version of this article (10.1186/s13104-017-3002-1) contains supplementary material, which is available to authorized users. Vulpes lagopus has a circumpolar distribution and inhabits all accessible Arctic tundra habitats . Fourteen of these were resighted as adults and only six bred in the study area. Although not all pups in the study area were tagged, this suggests a lack of natal philopatry .This study confirms the absence of an isolation-by-distance pattern but reveals cryptic genetic subdivisions in our population. As reported in other studies , 31, usiOne limitation of our study is that the study area may have been too small to detect a clear pattern of isolation-by-distance for a mobile species. Considering the relatively large distances between close relatives reported in Svalbard , foxes iAdditional file 1: Figure S1. Barplot of spatial principal component analysis (sPCA) eigenvalues and screeplot displaying each eigenvalue according to its variance and spatial autocorrelation (Moran’s I) components. Graphical results of the sPCA analysis of arctic foxes (n = 203) from Bylot Island, Nunavut, Canada.Additional file 2: Figure S2. Bayesian genetic structure analysis conducted with the software STRUCTURE. Graphical results of the Bayesian genetic structure analysis of arctic foxes (n = 203) from Bylot Island, Nunavut, Canada conducted with the software STRUCTURE.


We empirically investigated the effect of mental imagery on young children’s music compositional creativity. Children aged 5 to 8 years participated in two music composition sessions. In the control session, participants based their composition on a motif that they had created using a sequence of letter names. In the mental imagery session, participants were given a picture of an animal and instructed to imagine the animal’s sounds and movements, before incorporating what they had imagined into their composition. Six expert judges independently rated all music compositions on creativity based on subjective criteria . Reliability analyses indicated that the expert judges demonstrated a high level of agreement in their ratings. The mental imagery compositions received significantly higher creativity ratings by the expert judges than did the control compositions. These results provide evidence for the effectiveness of mental imagery in enhancing young children’s music compositional creativity. Eminent composers such as Mozart and Beethoven have been hailed as “geniuses”, a term first used in the 18 ability . Indeed, ability –4, and a ability , the Bri ability , and Sin ability .According to the “Four Ps” model of creativity –11, concStudies of the music compositional sketches of “geniuses” such as Mozart and Beethoven have dispelled the romanticised vision of creative products being conceived in singular, complete form through divine inspiration without the need for revision, while hinting at creative processes that are far less removed from those of ordinary individuals –25. ThatMental imagery involves a “quasi-perceptual experience: experience that significantly resembles perceptual experience (in any sense mode), but which occurs in the absence of appropriate external stimuli for the relevant perception” (p. 209 in ). For inWhile there may be modality-specific differences between our internal representations of visual and auditory stimuli , a singlWinnsboro Cotton Mill Blues, for example, the composer draws on visual and auditory imagery of a cotton mill to make parallels to the hammer mechanism of a piano, as well as the pianist’s mechanical hand and arm movements required for both a sonic representation of the mill’s industrial sounds and a visual-motor representation of repetitive physical labour. Indeed, one can easily identify a rich variety of extra-musical sources of composing inspiration that have lent themselves to visual and auditory imagery in programmatic music [The Carnival of the Animals conjures a “zoological fantasy” with the auditory sounds of a lion’s majestic roaring, a cuckoo’s gentle calling, and even the braying of donkeys. Similarly, Schubert’s The Trout piano quintet uses joyful, rippling arpeggios to recreate the vivid visual image of a fish’s lively movements in sparkling waters as it evades capture by a fisherman. As these examples illustrate, imagery may support the divergent exploration and discovery of novel, meaningful cross-modal associations between visual and auditory stimuli, as well as the development of subsequent creative musical ideas. Here, a fruitful parallel can be drawn to the literary domain, in which compelling colour-sound correspondence and visual-auditory metaphors  have ben green” .Accordingly, imagery could be highly useful in stimulating inspiration during the generation and exploration of musical ideas in the creative process, whereby composers consider various possibilities and engage in divergent thinking. In consonance with this notion, Antonietti  has discFurthermore, our mental representations are thought to be flexible and convertible, and thus amenable to manipulation . While sTaken together, it is possible that the manipulability of mental images may aid the creative synthesis of simple components into a useful object . In suppIndeed, the capacity to engage in visual and auditory imagery has been considered a component of creative musical aptitude in Webster’s  Measure Despite the theoretical utility of mental imagery and its plausible conceptual association with music compositional creativity, few studies to date have examined this relationship empirically. As Hubbard  has poinAlthough some studies have investigated composers’ use of mental imagery, these works often bear indirect links to compositional creativity as a key outcome of interest. For instance, while Kratus  exploredproduct can be evaluated based on consensual criteria in relation to a pool of artefacts being judged, since experts who possess domain knowledge and experience can reliably recognise creativity and arrive at a reasonable level of agreement in their subjective assessments of creativity even without the imposition of any external criteria. Under this view, a product is creative to the extent that the appropriate judges independently agree that it is creative. Accordingly, creativity in this study was operationalised as a novel and effective music composition as evaluated by expert judges using Amabile’s [Drawing on historical case studies of composers such as Saint-Saëns, we manipulated mental imagery use through instructing young child composers to imagine both the movements  and sounds (auditory imagery) of a given animal, before incorporating what they had imagined into their composition. To measure creativity, we adopted Amabile’s  Consensumabile’s  CAT, in mabile’s , 68–69. M = 6.85, SD = 1.36). As the MYC programme integrates music composition in its curriculum along with keyboard performance, all participants possessed prior experience in composition and had also received similar training related to music notation and basic compositional techniques. Participants were recruited after written informed consent from their parents and the music school’s Principal had been obtained. This study’s protocol was approved by the Institutional Review Board of the National University of Singapore.The participants were 9 children (6 were girls) enrolled in a special group-based music educational programme in Singapore , although one participant’s data were subsequently excluded from analyses due to non-conformance to instructions. Participants were 5- to 8-year-olds  were also included as check items. Participants rated the eight imagery objects’ imageability on a scale from 1  to 5 . The scale points were represented by black dots that increased in size by a constant ratio of 1:1.5 to aid participants in understanding the continuum.The children who participated in the pilot study reported how easy it was for them to imagine a given animal moving, as well as to imagine the sounds produced by the animal. Six animals were evaluated: bee , frog , and tiger . Accordingly, these animals served as the critical imagery items in the main study.Participants’ auditory and visual imageability ratings were collapsed to yield a mean imageability score for each animal. Data from four participants who did not fully understand the task, as indicated by their high mean imageability ratings above the mid-point of the scale for at least one of the two check items, were excluded from analyses. The three animals with the highest imageability scores were: The main study consisted of two phases: a composition phase, and a judging phase.Each participant underwent two music composition sessions comprising the mental imagery condition and control condition, respectively. Both sessions were conducted at least a week apart in counterbalanced order. Each session was conducted in groups of three to six participants, and lasted approximately 30 minutes. During the sessions, participants were allowed to freely try out their compositions on digital pianos. They were also given headphones to ensure that they could compose independently without external influence, and that they could not hear their peers’ compositions.In the control condition, participants were presented with the letter names of the seven white keys of a piano keyboard (from A through to G), and were instructed to write down any six letter names. Participants were told that this sequence of letters would constitute the motif of their composition, and that the letters could be used more than once. Following which, participants were asked to compose an 8-bar melody that sounded good to them based on the temporary motif that they had created, which could be modified in the course of composing. This compositional task was designed with reference to a traditional approach towards music composition, whereby composers create melodies through generating and exploring musical material based on a short motif  that lends itself to development . This teIn the mental imagery condition, participants were randomly assigned one of the three animals that had been selected via the pilot study. Participants were given handouts containing a picture of their assigned animal in a stationary position, and instructions for engaging in visual-auditory  mental imagery. Specifically, participants were asked to “hear” and imagine in their head the sounds made by their given animal, as well as to “see” and imagine the given animal moving. Following which, participants were instructed to compose an 8-bar melody that sounded good to them based on the animal they had imagined, such that listeners would be able to hear the animal “come alive” when listening to the composition. Imagery instructions were verbally reinforced for each individual participant to ensure understanding.After each composition session, all participants reported how easy it was for them to compose the piece of music, and how creative they perceived their composition to be. All ratings were performed on a 5-point scale, with points represented by black dots that increased in size by a constant ratio of 1:1.5 to illustrate the continuum. Participants were also asked to give their music composition a title, and to describe their composition. Responses to these check items were analysed to ensure that participants fully understood the imagery instructions given to them, and that their compositions were indeed about the animals that they had been told to imagine. With the exception of one participant who was subsequently excluded from analyses, all remaining 8 participants reported that they had adhered to the imagery instructions, and had written their imagery composition based on the sounds and movements of their assigned animal.Participants’ compositions were entered into a music notation software (Sibelius 6), and piano sound recordings were generated for each composition via the software’s playback function. The musical stimuli were then presented via a Microsoft Powerpoint slideshow to six expert judges. In accordance to Amabile’s  recommennot at all) to 7 (extremely). This item had been constructed based on Amabile’s [global  and implicit (deliberately little description of the rated dimension and criteria to consider) rating scales are best predictive for the construct of creativity. Following Amabile’s [The judges independently listened to and then rated each composition’s creativity  on a scale from 1  and control  conditions, t(7) = 1.18, p = .28. Participants did not perceive either composition task to be significantly easier or more difficult.To check that task difficulty was comparable between the mental imagery and control conditions, a paired samples Cronbach’s alpha was computed to test interjudge reliability, whereby a figure of .70 or higher was taken to indicate an acceptable level of agreement among the judges . The sixt-test revealed that the expert judges awarded significantly higher creativity ratings to the mental imagery compositions  than to the control compositions , t(7) = 2.30, p = .027, one-tailed, d = 0.81.Consistent with our hypothesis, a paired samples directional t-test indicated that the child composers perceived their own mental imagery compositions to be significantly more creative  than their control compositions , t(7) = 1.93, p = .048, one-tailed, d = 0.68, although the child composers’ creativity ratings did not significantly correlate with those by the expert judges, r(16) = -.38, p = .14. The child composers’ ratings of their compositions are presented in Echoing the expert judges’ ratings, a paired-samples directional We investigated the use of visual-auditory mental imagery in young children’s music compositional processes, and established empirical support for its positive effects on music compositional creativity. As assessed by a panel of expert judges using Amabile’s  CAT, theAlthough some indirect support for the creative benefits of mental imagery has previously been accrued in meta-analytic research and correlational studies that examined the relationship between visual imagery abilities and divergent thinking performance –77, it mAt the same time, our findings corroborate Piagetian developmental theories that children as young as 5 years of age  are capable of engaging in mental imagery, and are able to use their mental images to form symbolic representations in the absence of actual perception –81. AccoFurthermore, the creative advantage of employing mental imagery emerged even though the children rated the imagery composition task as being of similar difficulty as the control composition task, with mean ratings around the midpoint of the scale. That both tasks were perceived to be of comparable difficulty (or ease) is, perhaps, unsurprising from a developmental perspective. Just as how the use of mental imagery has been reported to be evident even in preschoolers’ early make-believe play , the traThese results have promising implications for music composition pedagogy, whereby music educators can incorporate mental imagery techniques in their teaching of composition to nurture their students’ creativity. A particular strength of the mental imagery strategy is that it appears to be relatively easy to implement in the classroom—the child composers in the present sample profited from mental imagery even without training prior to completing the experimental task, with the benefits of mental imagery for their creative performance emerging immediately. As younger children often tend not to spontaneously use mental imagery—particularly visual imagery—in problem-solving although they are capable of doing so –85, theyWith experience and the development of domain-relevant skills, knowledge, and expertise over time, composers may become more proficient at mentally manipulating and organising musical material through the use of imagery, and may also be more adept at applying diverse compositional techniques to effectively “translate” their extra-musical images and fully express their creative ideas , 86. ForThis study represents an initial foray into the relationship between the use of mental imagery and music compositional creativity, targeting a select group of young students who have received music compositional training under the special MYC programme. One limitation of this study is hence its small sample, although it is worth noting that we observed medium to large effect sizes in spite of this. Further research could validate and extend our findings by involving larger samples of general music learners and composers.In particular, future work can investigate and identify the specific ways in which young children use mental imagery to enhance music compositional creativity. Although the child composers received similar instructions to imagine the sounds and movements of a given animal, each individual may have applied the resulting mental images in a different way, or during a different stage in the compositional process. For instance, whereas some children in this study attempted to directly translate their mental images into musical contexts , others seemed to use their images as a broader concept or mood that shaped their final compositional product . Subsequent studies can thus qualitatively probe young children’s music compositional processes to gain deeper insight about the nature of their mental imagery and the ways in which it is applied, as well as map the developmental trajectory of imagery use in music composition in view of age-related differences in image generation and maintenance abilities .flower and snowman even though these two objects presumably could not move or produce sounds on their own accord. Upon consideration of young children’s ability to engage in highly imaginative make-believe play [At the same time, future studies can extend the effects of mental imagery on music compositional creativity to more diverse imagery targets besides the animals used in the present research. In our pilot study, some children reported high imageability for a eve play , exploriFurthermore, given the prevalence of multisensory imagery in everyday cognition –92, it mRelatedly, the qualities associated with music compositions that are perceived as “creative” are not readily inferred from the present study, since the CAT crucially depends on subjective global evaluations of creativity and does not ask raters to justify their ratings . Our anaIn addition, since this study has established that music compositional creativity can be promoted through the use of mental imagery as a cognitive strategy, a meaningful avenue for future work is to explore more techniques to increase young children’s repertoire of approaches towards creatively composing music. For instance, some studies with seventh-and ninth-grade students have demonstrated that music compositions that had been notated using non-traditional graphic patterns were rated as aurally more creative by expert judges, as compared to compositions that had been written using traditional staff notation –96. PresWhile musical creativity has often been associated with “geniuses” such as Mozart and Beethoven, this research demystifies creativity by providing suggestive evidence that it can potentially be nurtured in ordinary individuals through essentially ordinary cognitive processes that yield extraordinary products . The useS1 Dataset(XLSX)Click here for additional data file.


Onychoprion fuscatus. An urgent call to re-assess their conservation status has been made given that some populations, such as the one on Ascension Island, South Atlantic, have declined by over 80% in three generations. Little is known about their population genetics, which would aid conservation management through understanding ecological processes and vulnerability to environmental change. We developed a multiplex microsatellite marker set for sooty terns including sex-typing markers to assist population genetics studies.Seabirds have suffered dramatic population declines in recent decades with one such species being the sooty tern Fifty microsatellite loci were isolated and tested in 23 individuals from Ascension Island. Thirty-one were polymorphic and displayed between 4 and 20 alleles. Three loci were Z-linked and two autosomal loci deviated from Hardy–Weinberg equilibrium. The remaining 26 autosomal loci together with three sex-typing makers were optimised in seven polymerase chain reaction plexes. These 26 highly polymorphic markers will be useful for understanding genetic structure of the Ascension Island population and the species as a whole. Combining these with recently developed microsatellite markers isolated from Indian Ocean birds will allow for assessment of global population structure and genetic diversity.The online version of this article (10.1186/s13104-017-3084-9) contains supplementary material, which is available to authorized users. Onychoprion fuscatus are long-lived pelagic seabirds distributed throughout the tropical oceans where their range covers in excess of 20,000 km2 [Sooty terns ,000 km2 . They ar,000 km2 , make it,000 km2 . Declini,000 km2 .The sooty tern population on Ascension Island declined by 84% between 1942 and 2005 . A recenSince 2000 the breeding population on Ascension Island has occupied two main areas at Mars Bay and Waterside that are approx. 3 km apart Fig. . Fine-scGenomic DNA was extracted from blood samples using an ammonium acetate precipitation method , 9. Micr2O and 1 μl of this solution was added to 9 μl formamide and 0.2 μl of 500-ROX size standard . An ABI 3730 DNA Analyser was used to separate PCR products and alleles were scored using GENEMAPPER v 3.7 . Of the 50 primer sets tested, 14 either did not amplify or produced a non-specific product, five loci were monomorphic and three were Z-linked  on Ascension Island . The 26 autosomal microsatellite loci, together with the three sex-typing markers, were combined into seven plexes by inclusion of the fluorescent dye NED , to create a multiplex marker set using Multiplex Manager 1.2 [HO) and expected heterozygosities (HE) were 0.82 ± 0.12 and 0.82 ± 0.09, respectively. The probability of identity for the 26 loci was 4.1 × 10−33.To ensure allele frequencies were not biased by over-representation of genotypes through inclusion of related individuals , pairwisEX v 6.5 , 30. Twoager 1.2  and valiThis multiplex set containing a large number of novel microsatellite loci together with the three sex-typing markers will be of great utility for fine- and large-scale population genetic structure analyses. More specifically, this multiplex set offers an effective and economical approach for investigating parentage assignment, relatedness and assisting conservation management plans for this colonial seabird. Combining this multiplex set with 16 microsatellite markers recently developed for sooty terns in the Indian Ocean  would alAnous stolidus) and black noddies (A. minutus) still needs to be assessed.The present study was carried out independently of the study by Danckwerts et al.  but ongo