Bemisia tabaci (Hemiptera: Aleyrodidae) is a cryptic species complex composed of more than 34 putative species. Moreover to the primary endosymbiont Portiera aleyrodidarum, six secondary endosymbionts Cardinium, Arsenophonus, Rickettsia, Wolbachia, Hamiltonella and Fritschea are known in B. tabaci. Here, we tested four of the six secondary endosymbiont lineages (excluding Fritschea and Hamiltonella) from 180 whitely individuals collected from six host plants belonging to families Solanaceae  and Fabaceae . Phylogenetic studies grounded on the mitochondrial cytochrome I gene revealed the presence of Asia 1, Asia II 1 and Asia II 7 genetic groups for B. tabaci. Specific primers targeting 16S rRNA and 23S rRNA gene were used for estimating the bacterial endosymbionts. As a primary endosymbiont Portiera aleyrodidarum was present in all the studied samples; whereas, an uneven distribution of secondary endosymbionts were recorded. Overall our finding exposes the variation and diversity of endosymbionts within the B. tabaci collected from different host plants and outlines the genetic groups of the insect pest. The study delivers a significant information concerning the circulation of secondary endosymbionts with host preferences of B. tabaci and provides suggestion for progressive studies on targeting the specific endosymbionts with respect to host for the control measures.Endosymbionts are vital factor for arthropod ecology. The whitefly  Bemisia tabaci (Hemiptera: Aleyrodoidea) is a wide-reaching pest and has triggered substantial destruction to legumes, flowers, vegetables, grains and cotton production through direct nourishing, emitting honeydew, bringing host plant phytotoxic disorders, and communicating more than 120 plant viruses. , whB. tabaci as prior demarcated by the researchers [An investigation of individuals from solanaceous and fabaceous hosts revealed a divergence in the distribution frequency of secondary endosymbionts with host preferences and genetic groups. This study recommends the relation among the symbiotic bacterial populations and the genetic groups of earchers ,29,39. Tearchers .viz., increasing the range of temperature tolerance [Insects are identified for having endosymbiotic microorganisms producing a diversity of symbiotic relations stretching from mutualism to parasitism ,41. The olerance , enhanceolerance  and possolerance .Cardinium was higher in individuals belonging to Asia II 1 genetic group. As the results describes the percentage presence of Cardinium in tomato, potato, soyabean and mungbean individuals were 100%, 93.33%, 76.67% and 96.67% respectively. The lowest presence of Cardinium was noted in Asia II 7 genetic group , followed by Asia 1 genetic group . For Rickettsia percentage presence was higher in Asia 1 genetic group , whereas an average percentage distribution was recorded in Asia II 1 and Asia II 7 genetic groups , whereas in Asia II 1 genetic group it was found in least proportion as the potato harbors 10%, soyabean and mungbean harbors 3.33% and interestingly it was totally absent in tomato individuals. The percentage distribution of Wolbachia was 16.66% in Asia II 7 genetic group (subabool). The distribution frequency of Arsenophonus was recorded higher in subabool individuals as 90% belonging to Asia II 7 genetic group. Whereas, the percentage presence of Arsenophonus was least recorded in the brinjal individuals as 16.66% belonging to Asia 1 genetic group. These outcomes are in contract with the earlier reviews by Chiel et al. (2007), Gueguen et al. (2010) and Gnankine´ et al. (2013) [Hamiltonella and rarely low frequencies of Wolbachia and Cardinium, while Q 3 individuals harbour commonly Arsenophonus with a high level of co-infection with Rickettsia. In added words, there is an inequality in the distribution of secondary endosymbionts inside varied genetic groups of B. tabaci.The percentage distribution of c groups . In case. (2013) ,29,39 whB. tabaci in New Delhi, India. The results point out an inevitability for groundbreaking revisions on the host wise occurrence of secondary endosymbionts and its link with different genetic groups of B. tabaci and their impact in the polyphagous nature of this insect pest.The study is trying to document the distribution incidence the secondary endosymbionts related with host's preference of Bemisia tabaci samples were collected from fields of the Indian Agricultural Research Institute, New Delhi, during 2016 and 2017, and preserved particularly in Eppendorf tubes with absolute alcohol at -200C until further handling. Sample size of 180 individuals from six hosts of two families were used for the study. Whole genomic DNA of each adult single fly was retrieve by means of DNASure Tissue Mini Kit  as per manufacturer's protocol and kept at -20°C. The extracted DNA was used in the succeeding experiments.B. tabaci was concluded aground on mitochondrial cytochrome oxidase I (mtCOI), subsequently a PCR reaction with universal primers [The identification of the genetic group of  primers  (Table 1https://blast.ncbi.nlm.nih.gov/Blast.cgi). The genetic group uniqueness was additionally recognized by the phylogenetic and molecular evolutionary analysis with well-assigned homologous sequences of the B. tabaci genetic groups from the consent sequence databank by MEGA version 6 [36 and multiple sequence alignments were attained with the Clustal W37 algorithm using default parameters. The sequences were submitted to NCBI for GenBank Accessions . Statistical assessments were completed with SPSS version 16.0.The divergences in comparative number of endosymbionts in B. tabaci on Solanaceae and Fabaceae host plants of New Delhi, India. The consequences originated an agreement that there is a breach in the facts of existence of secondary endosymbionts with respect to the host plants and genetic groups; and suggests an obligation for lenient enhancements on the host wise occurrence of secondary endosymbionts and its stretch with numerous genetic groups. A broadminded and proportionate survey is obligatory to reveal the confirmations concerning the role of these endosymbionts and the source of uneven passage frequency of these secondary endosymbionts.The present study was concentrated on finding the endosymbiont array associated with


Genotyping of highly polymorphic autosomal short tandem repeat (STR) markers is a potent tool for elucidating genetic diversity. In the present study, fifteen autosomal STR markers were analyzed in unrelated healthy male Gorkha individuals (n = 98) serving in the Indian Army by using AmpFlSTR Identifiler Plus PCR Amplification Kit. In total, 138 alleles were observed with corresponding allele frequencies ranging from 0.005 to 0.469. The studied loci were in Hardy-Weinberg Equilibrium (HWE). Heterozygosity ranged from 0.602 to 0.867. The most polymorphic locus was Fibrinogen Alpha (FGA) chain which was also the most discriminating locus as expected. Neighbor Joining (NJ) tree and principal component analysis (PCA) plot clustered the Gorkhas with those of Nepal and other Tibeto-Burman population while lowlander Indian population formed separate cluster substantiating the closeness of the Gorkhas with the Tibeto-Burman linguistic phyla. Furthermore, the dataset of STR markers obtained in the study presents a valuable information source of STR DNA profiles from personnel for usage in disaster victim identification in military exigencies and adds to the Indian database of military soldiers and military hospital repository. Gorkha  soldiers are a dominant force in the Indian Army who have completed 200 years in the Armed Forces since their integration on April 24, 1815. They are courageous in battle and have won many gallantry awards and military honors. Gorkha was the usual designation of the reigning dynasty of Nepaul Gurkha’ and ‘Gurkhali’ are synonymous with ‘Nepali’ and were derived from the name of old kingdom of Gorkha (Gurkha), a hill town and present day district of Gorkha (~fifty kilometers west of Kathmandu) from which the kingdom of Nepal expandedThe Only a handful of published genetic studies on Gorkhas/Nepalese are available567891011Nepalese population displays strong genetic differentiation despite sharing close geographical proximity underscoring necessity for studies of Nepalese ethnic groups. The understanding of past genetic history, genetic affinity as well as population diversity has been vastly improved by the molecular genetic markers like microsatellite, mitochondrial DNA and Y chromosome markers. Microsatellite markers, particularly the autosomal short tandem repeats (STR), which have high polymorphic information content (PIC) and high power of discriminationTPOX locus had the maximum allele frequency with allele 8 (0.469) being the most frequent allele in the population. Observed heterozygosity of TPOX, TH01 and D5S818 was found to be low being 0.602, 0.663 and 0.663 respectively. Remaining STR loci were highly polymorphic with observed heterozygosity values ranging from 0.724 for CSF1PO to 0.867 for D18S51. All loci met HWE expectations. Allele frequencies and summary statistics of the 15 autosomal STRs in the studied Gorkhas are presented in TPOX) to 0.87 (FGA) and power of discrimination (PD) ranged from a minimum of 0.843 for TPOX to a maximum of 0.963 for FGA. The most polymorphic locus of FGA was also, as was expected, the most discriminating in the population. The power of exclusion (PE) ranged from a minimum of 0.293 for TPOX to a maximum of 0.770 for D21S11 and D19S433. The combined power of discrimination (CPD) and combined power of exclusion (CPE) for all 15 STR loci was 0.9999999986 and 0.9999993441 respectively. The average probability of matching value was found to be 0.082 and it was expressed as 1 in 14.7.In total, 138 alleles were observed in the studied population with allele frequencies ranging from 0.005 to 0.469. A distance matrix is presented in 8Phylogenetic analysis based on allelic frequencies was performed to investigate genetic relationship of the studied population with the neighboring and Indian lowlander population8910161718192021The present study reveals phylogenetic relationship of Gorkhas of Tibeto-Mongol origin with neighboring Himalayan population. Our findings are in concordance with the previous studies on Indian Gorkhas which suggested genetic closeness of Gorkha population with that of Mongoloid origin based on HLA-A and B antigensGeographical continuity is a major influencing factor of genetic affinity among diverse populations which are distributed over a wide geographical area after migration. Geographically adjoining populations from a common stock cluster together due to genetic affinity and the findings of the present study with autosomal STR markers substantiate the genetic affinity of the Gorkhas with the Tibeto-Burman linguistic phyla reflecting recent past genetic history and possible migration from Tibet as well as probable origin of the Gorkhas from Mongolian and/or Tibetan stocks. Studies with Y- chromosomal diversity revealed high frequency of East Asian specific haplogroup O3a5-M1324 in the Himalayan population of Nepal and Tibet suggesting a common ancestry for these linguistic subfamilies8It was observed that our study population comprised both Tamangic  and Magaric (Magar) groups of Tibeto-Burman language family, based on assessment of ethnicity from ethno-linguistic questionnaire . Further9India is home to various social groups with diverse ethnic and linguistic origins representing several racial stocks and social statuses that found place for themselves at various points of time and adapted to several ecological niches offered by the physiographic and climatic setting of the area. The waves of migration drew the ancestors of the majority of the present population of the area from the surrounding territories and across the Himalayas37383940One of the other objectives of our study was to create a microsatellite dataset of Indian soldiers for human identification for military purposes. Military exigencies and major disasters such as wars, airplane accidents and maritime transport disasters leave military personnel highly vulnerable. Usage of weapons of mass destruction and terrorist action also expose the military to disaster and fatality. Identification of remains brings sense of comfort and closure to family and friends. The STR markers are important tools for human identification18The present study reveals phylogenetic relationship of Gorkhas of Tibeto-Mongol origin with other neighboring Tibeto-Mongol Himalayan population. The study substantiates genetic affinity of the Gorkhas with the Tibeto-Burman linguistic phyla reflecting recent past genetic history and origin of the Gorkhas from Mongolian and/or Tibetan stocks. Furthermore, the dataset of STR markers obtained in this study presents a valuable information source of STR DNA profiles from military personnel for usage in disaster victim identification in military exigencies and adds to the Indian database of military soldiers and military hospital repository.The study and the experimental protocols were approved by the Ethics Committee of the Defence Institute of Physiology and Allied Sciences, Delhi. The participants (n = 100) were selected from the Gorkha regiment of the Indian Army who were part of an ongoing study of the Institute. All participants gave written informed consent before enrolment in the study. The experiments were conducted in accordance with quality control measures at the Department of Molecular Biology of Defence Institute of Physiology and Allied Sciences, Delhi, Ministry of Defence, India which is an accredited laboratory (ISO 9001:2008).2 EDTA vacutainers  and stored at −20 °C till further processing.Of the participants who reported, 59 individuals were from Nepal and 41 from India. During the study period, they were stationed at sea level. The clan ties were determined based on self-reporting and ethnic backgrounds were ascertained through ethno-linguistic questionnaire. To ensure that the individuals were ethnically unmixed, both parents had to belong to the same group. 3–4 ml of peripheral blood was collected through venepuncture from the consenting individuals in K260/A280 ratio of 1.8–1.9. Further quantification of DNA was performed by the Quantifiler Duo Human DNA Quantification kit  using 7500 Real Time PCR system  with v1.1 software as per manufacturer’s protocol.High molecular genomic DNA was extracted as per published procedureD8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) along with the gender determination marker Amelogenin with AmpFlSTR® Identifiler® Plus kit  on GeneAmp PCR system 9700 Thermal Cycler following manufacturer’s recommended protocol. 2 samples failed in amplification. Positive and negative amplification controls were used as per kit guidelines. The amplified products were run on 3500xL Genetic Analyzer  using 36 cm capillary array and Dye set G5. Allelic ladder sample provided in the kit was included in each run. Data generated using capillary electrophoresis was analyzed using Gene Mapper ID-X version 1.4 software  as per manufacturer’s instructions. Allele calls were generated for all samples and exported in Excel format. Plot views were generated in PDF format.Polymerase chain reaction (PCR) amplification was carried out for each sample using 15 autosomal STR loci markers (obs) and expected heterozygosity (Hexp) and Hardy-Weinberg Equilibrium (HWE). HWE based on the exact test was confirmed for all the studied 15 loci at a significance level of p >0.003 after Bonferroni correctionAllelic frequencies for the 15 STR loci and matching probability (Pm), power of discrimination (PD), power of exclusion (PE) and polymorphic information content (PIC) were computed using the PowerStats version 1.2 spreadsheet programHow to cite this article: Preet, K. et al. Genetic diversity in Gorkhas: An autosomal STR study. Sci. Rep.6, 32494; doi: 10.1038/srep32494 (2016).


Diplodus vulgaris, a coastal fish that is both commercially and ecologically important. Adults were sampled in 7 locations (stretches of coastline approximately 8 km long) and juveniles in 14 sites (~100 to 200 m of coastline) along 200 km of the Apulian Adriatic coast (SW Adriatic Sea), within and outside an MPA . Our study found similar genetic diversity indices for both the MPA and the surrounding fished areas. An overall lack of genetic structure among samples suggests high gene flow (i.e. connectivity) across a scale of at least 200 km. However, some local genetic divergences found in two locations demonstrate some heterogeneity in processes renewing the population along the Apulian Adriatic coast. Furthermore, two sites appeared genetically divergent, reinforcing our observations within the genetic makeup of adults and confirming heterogeneity in early stage genetics that can come from either different supply populations or from chaotic genetic patchiness occurring under temporal variation in recruitment and in the reproductive success. While the specific role of the MPA is not entirely known in this case, these results confirm the presence of regional processes and the key role of connectivity in maintaining the local population supply.Patterns of connectivity and self-recruitment are recognized as key factors shaping the dynamics of marine populations. Connectivity is also essential for maintaining and restoring natural ecological processes with genetic diversity contributing to the adaptation and persistence of any species in the face of global disturbances. Estimates of connectivity are crucial to inform the design of both marine protected areas (MPAs) and MPA networks. Among several approaches, genetic structure is frequently used as a proxy for patterns of connectivity. Using 8 microsatellite loci, we investigated genetic structure of the two-banded sea bream  Levels of self-recruitment within and connectivity among populations are key factors influencing marine population persistence and stock sustainability , as wellPopulation genetics has been and remain the most widely used approach to indirectly evaluate connectivity from genetic structure (i.e. heterogeneity) among populations in marine organisms , 19 assuDiplodus vulgaris  as a model species and Torre Guaceto  as a model MPA. We selected the two-banded sea bream because of its positive response to MPA in terms of biomass recovery [2 [D. vulgaris populations across the Almeria-Oran front in the western Mediterranean. Whereas, other studies conducted on its congeneric species D. sargus showed weak but significant genetic differences for populations at a scale ranging from less than 100 km [In this study, we investigated patterns of connectivity between an MPA and surrounding fished areas using the two-banded sea bream recovery , 27 and recovery –30. Thisrecovery , 31. It recovery  and adulovery [2 , 34. Limovery [2 . Galarzaovery [2  showed sn 100 km  to severn 100 km .D. vulgaris along about 200 km of coast encompassing an MPA and fished areas, and 2) to estimate the amount of immigrants and self-recruitment in TGMPA, using assignment tests [The TGMPA (SW Adriatic Sea) was adopted as a model MPA because of its effective enforcement since 2001 and because previous studies showed that fishing bans within its no-take zones allowed for the recovery of both fish , 40 and nt tests –43.To assess the observed genetic differentiation at a local scale, 7 locations (i.e. stretches of coastline approximately 8 km long) were selected starting from TGMPA, spanning both northward and southward, up to about 100 km from the MPA borders .Overall, 525 adults from 7 locations were sampled from April 2010 to July 2011 . In conjet al. [Microsatellite loci were optimized from Roques et al. , 45 and STAT v. 2.9.3 [Traditional genetic statistics were performed using Fv. 2.9.3  to descrv. 2.9.3 .The data were tested for the presence of null-alleles using MICROCHECKER v. 2.2.3 . DeviatiSTATISTICS were computed through an analysis of molecular variance (AMOVA) performed in GENALEX v. 6.502 [Fv. 6.502 . Genotypv. 6.502  for multv. 6.502  were alsFst measures were graphically represented through multidimensional scaling (MDS) using PRIMER 5 v.5.2.9. This method computes coordinates for each sample such as the distance between points fitted to the measured distances between populations. The accuracy of the data was measured with a stress factor [Pairwise s factor .et al. [In parallel, STRUCTURE 2.3.1 , 54, 55 et al. . Four ruet al.  as impleet al. .P > 0.5) it was left as unassigned.Juveniles were assigned to the 7 putative adult samples based on their multi-locus genotype probabilities using a Bayesian assignment method . This meFis, All loci appeared to be moderately to highly polymorphic, exhibiting 7–28 alleles per locus . AllelicP > 0.5). Allelic richness (Ar) ranged from 13.9 to 15.27, while He and Ho ranged from 0.79 to 0.81 and from 0.70 to 0.75, respectively.Genetic diversity indices appeared quite homogeneous among adult samples  with no Fst equal to 0.006 (P < 0.001). Levels of differentiation, as measured by pairwise Fst, between pairs of samples were low  and all other samples with values ranging from 0.0140 to 0.0256  and the MPA (TG) with an Fst = 0.0084  with Torre a Mare (TAM), Punta Penne (PP) and Torre Pozzella (TP) locations showing the lowest values (Allelic richness (Ar) ranged from 13.37 to 15.12. He and Ho ranged from 0.77 to 0.83 and from 0.72 to 0.83, respectively . Signifit values .Fst equal to 0.011 (P < 0.001). Levels of differentiation were low but significant in numerous pairwise comparisons once a sequential Bonferroni adjustment was applied  sampled with similar proportions of membership to each cluster with the exception of individuals sampled at site 12 (PP) and 13 (TAM) . These jFst equal to 0.015 (P < 0.001). Levels of differentiation, as measured by pairwise Fst, were low but significant between most of the samples An AMOVA was used in an analysis of all individuals sampled, for both adults and juveniles, to explore how juveniles may segregate with respect to adults. The analysis showed that 1% of the genetic variation was explained by genetic differences among the pooled individuals with an overall The MDS two-dimensional plot showed an overall grouping of all individuals with the exception of juveniles from Punta Penne (PP), Torre a Mare (TAM), and adults from Monopoli (M). Such observations were similar to those previously conducted where juveniles and adults were analyzed separately.A total of 107 juveniles out of 755 (14%) were assigned to one of the adult samples. Most of these juveniles were assigned to Monopoli (M) or Torre Guaceto (TGMPA) adult samples . Conversely, 62% juveniles were left unassigned because they were assigned to more than one adult sample .A total of 179 juveniles out of 755 (24%) had a probability lower than 0.05 of belonging to any sample and were designated as being immigrants coming from genetically distinct populations than those of the adults in our study area. Therefore, the remaining 76% of juveniles collected in the 14 sites originated from adults collected in the 7 locations along the 200 km of the study area.Regarding TGMPA, 24% of juveniles (23 out of 94) are immigrants. Twelve % of the juveniles collected in TGMPA  are origDiplodus vulgaris along ~ 200 km of the Apulian Adriatic coast (SW Adriatic Sea) did not reveal any structured pattern attributable to the presence of TGMPA with respect to distance. However, we found local genetic heterogeneity for juveniles from Punta Penne (PP) and Torre a Mare (TAM); and for adults from Monopoli (M), resulting in genetically differentiated entities. In addition, we demonstrate that 12% of juveniles sampled in TGMPA are originated from adults in the surrounding fished areas, while 3% of juveniles sampled outside TGMPA are originated inside. On the other hand, about 24% of juveniles collected in the 14 sites located along the 200 km of coast originate from outside the surveyed area, where they display genetic combinations that we would not expect to find within the adults sampled from the study area. These results demonstrate patterns of connectivity in relation to the local MPAThe genetic structure analysis of the two-banded sea bream Diplodus species spawn in group [Looking at the quality of the dataset, some of the loci analyzed in this study showed a deviation from expectations for HWE with a significant deficit of heterozygotes in some sampling locations. Such a deficit may be due to various processes such as inbreeding, assortative mating, Wahlund effect  or null in group  and therDiplodus sargus. The genetic similarity indicates that at the scale of this study, gene flow is sufficient to maintain homogeneity in allele frequencies between TGMPA and the surrounding fished grounds along the sampling area (i.e. 200 km). The observed genetic similarity results from the recent isolation of the Adriatic population following the last sea level rise and continuous dispersion by sea currents dominating the western Adriatic [D. vulgaris spawning period (i.e. mainly winter) and the exchanges and potentially extensive dispersal at juvenile stage [No differentiation was found in the genetic diversity indices among the adult samples throughout the 200 km range within the South West Adriatic Sea. This result indicates that TGMPA is not an isolated protected location and that there is a patchwork of genetic connectivity among sampled locations. This result is in agreement with the study of Pujolar et al.  that shoAdriatic  during tle stage .Overall, little evidence of genetic structure was found among the 7 adult samples. Only the adults sampled in Monopoli appeared genetically different from all the others (except samples of TGMPA). In the meantime Monopoli samples show the lowest values of genetic diversity indices . The isolate genetic divergence of Monopoli samples suggest for some local process often enrolled into the concept of chaotic genetic patchiness. The temporal variability in the sampling could be a possible explanation for this divergence as the sampling occurred between 2010 and 2011 for all locations except for Monopoli which occurred entirely in 2011. The differentiation of the Monopoli population could be due to post-settlement natural selection events  since diD. vulgaris of up to 165 km was reported. These authors also highlighted the existence of three major natal sources of propagules responsible for the replenishment of their study area (i.e. 180 km of coastline), suggesting that propagule dispersal extends to at least 90 km [Overall, high values of genetic diversity and little evidence of genetic structure were found for juvenile samples. These results are most likely due to significant connectivity and admixture among the 14 sampling sites collected within a fairly limited spatial scale (maximum of 200 km apart) as well as habitat continuity along the sampling area. These findings are also consistent with the findings by Di Franco et al.  where exst 90 km .Fst values were low . The pairwise Fst analysis and the Bayesian method both demonstrated that some juveniles sampled in TAM and PP were genetically different from all other samples. TAM and PP samples showed the lowest genetic diversity indices  among all samples. This observed differentiation most likely originates from genetic drift linked to a reproduction event involving a limited number of adults. In fact, the localized heterogeneity found in the genetic constitution of some juveniles sampled could be interpreted as the result of a large variance in the local reproductive success [Some of the juvenile sites (e.g. TAM and PP) appeared significantly different from others although  success . A high  success . In addi success –71 obser success . This is success  which su success , 74, tha success .The lack of high genetic structure across the study area reflects the presence of significant gene flow within the studied distribution range. Conversely, and within the context of global homogeneity, the genetic patchiness found at some sites and locations reflects temporal variation in recruitment and variance of local reproductive success.D. vulgaris’ spawning period (i.e. mainly winter) is dominated by a multitude of turbulence and gyres at different spatial scales [The assignment method performed by GeneClass2 has the advantage taking into account the possibility of not having sampled all potential populations . Using tl scales . Therefol scales . On the Fst and STRUCTURE results.Regarding TGMPA, 12% of juveniles sampled inside the MPA are originated from adults in the surrounding fished areas, while 3% of juveniles sampled outside TGMPA are originated inside. In addition, 24% of juveniles sampled in TGMPA were found to be immigrants. Therefore, these results demonstrate patterns of connectivity in relation to the local MPA. The estimation of self-recruitment (3%) in TGMPA based on assignment tests should be treated with caution since low genetic differentiation was found in the Diplodus vulgaris populations is maintaining the local population supply. Measuring gene flow of D. vulgaris by genetic markers has allowed us to elucidate the distance of effective larval dispersal of this species in the study area.This study showed that TGMPA is part of an interconnected system and that the high gene flow among The spatial scale of the sampling outside the MPA cannot account for the entire dispersal kernel of individuals produced in the MPA making some limitation of the present work. However, recent paper that provided kernel of larval dispersal , 77 demoEstimates of larval dispersal distances can subsequently be used to optimize MPA placing within a network since an MPA has been proposed in the area of Otranto-Tricase (50 km south of San Andrea (SA)). This new MPA may improve the potential connectivity between TGMPA in the Adriatic and in Porto Cesareo MPA in the Ionian Sea. Thus, the results may serve as a baseline for future studies looking into the design and implementation of a potential network between TGMPA, Otranto-Tricase and Porto Cesareo MPAs.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableThe original primers were developed in Roques et al. , 45.(PDF)Click here for additional data file.S4 TableSee (PDF)Click here for additional data file.S5 TableBarplot graphical display of STRUCTURE analysis for K = 11 clusters based on Evanno’s ΔK. Each juvenile individual was represented by a vertical bar. Each color represents the relative membership proportion of each juvenile to each of the 11 clusters.(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S1 FigEach adult individual was represented by a vertical bar. Each color represents the relative membership proportion of each adult to each of the 3 clusters.(TIF)Click here for additional data file.S2 FigSee (TIF)Click here for additional data file.


IBD) is one of the main modes of differentiation in marine species, above all in species presenting low dispersal abilities. This study reports the genetic structuring in the tropical hydrozoan Macrorhynchia phoenicea α , a brooding species, from 30 sampling sites in the Western Indian Ocean and the Tropical Southwestern Pacific, using 15 microsatellite loci. At the local scale, genet dispersal relied on asexual propagation at short distance, which was not found at larger scales. Considering one representative per clone, significant positive FIS values (from −0.327*** to 0.411***) were found within almost all sites. Gene flow was extremely low at all spatial scales, among sites within islands (<10 km distance) and among islands , with significant pairwise FST values (from 0.035*** to 0.645***). A general pattern of IBD was found at the Indo‐Pacific scale, but also within ecoregions in the Western Indian Ocean province. Clustering and network analyses identified each island as a potential independent population, while analysis of molecular variance indicated that population genetic differentiation was significant at small (within island) and intermediate (among islands within province) spatial scales. As shown by this species, a brooding life cycle might be corollary of the high population differentiation found in some coastal marine species, thwarting regular dispersal at distances more than a few kilometers and probably leading to high cryptic diversity, each island housing independent evolutionary lineages.Assessing population connectivity is necessary to construct effective marine protected areas. This connectivity depends, among other parameters, inherently on species dispersal capacities. Isolation by distance ( First, the geographic origin of individuals  was treated as an a priori defined population, except in clustering analyses. Pairwise  Wright,  comparis Wright, ; the sig D Jost,  comparis D Jost, ; the siges Table . This rees Table . All Manes Table  with 1042.5adegenet  using the package Jombart,  in R v.3Jombart, . DAPC isJombart,  and thatJombart, ; we testK populations, and individuals are assigned probabilistically to each population in order to maximize HWE and minimize LD. Due to the important size of our dataset and following the recommendations of Rosenberg et al. (K (K varying from 1 to 10), three independent runs were conducted with a burn‐in period of 5 × 104 steps followed by 5 × 105 Markov chain Monte Carlo iterations. We used the statistic proposed by Evanno, Regnaut, and Goudet  and the Shared Allele Distance index (SAD). RD has been developed from the Goldstein distance index. It provides a parsimonious representation of the genetic distance between individuals based on the difference of the microsatellites allele lengths . The GD groups sites considering their historical origin, while FST takes into account the site structure. Once the matrices of genetic distances between individuals or sites were estimated, different networks were built considering individuals/sites and genetic distances as nodes and links between them, respectively. For the network construction, links were included for all distances and were removed in decreasing order until the percolation threshold (Dpe) was reached  and 33.1R ranged from 0.449 to 1.Using the 15 loci that amplified correctly (see Section 3.2SE) = 14.8 ± 1.7], with some loci monomorphic in several sites . However, nearly 50% of the positive tests  occurred due to monoallelic loci in various sites and might just reflect their general low genetic diversity. Observed heterozygosities ranged from 0.264 to 0.605 (mean ± SE = 0.392 ± 0.011) in JUA1 and MAD1, respectively, and unbiased expected heterozygosities from 0.287 to 0.610 (mean ± SE = 0.468 ± 0.015) in GDT9 and MAY2, respectively. Mean allelic richness per locus ranged from 1.729 ± 0.151 in GDT9 to 3.311 ± 0.399 in CHE6 and mean number of private allele per locus ranged from 0.005 ± 0.003 in GDT4 to 0.314 ± 0.165 in JUA1. Multilocus FIS values ranged from −0.327*** for MAD1 to 0.411*** for CHE4  to 27 (Mp24) [mean ± standard error (es Table . Signifi3.3K = 3 with two clusters corresponding to Grande Terre (GDT1 to GDT9), whereas MLGs from the Loyalty Islands (LOY1 to LOY6) were poorly assigned to two clusters  and some admixture , DAPC identified a clear distinction between MLGs from Western Indian Ocean and the Tropical Southwestern Pacific  at the percolation threshold (Dpe = 0.18) revealed strong relationships among sites from the same island/archipelago, especially in the New Caledonia ecoregion  showed that individuals of one island/archipelago remained linked and were more closely related to each other than to individuals from other islands/archipelagoes. In contrast, the network built with the RD index (Dpe = 5.62) resulted in less geographic structure among MLGs Fig. . The aven Figure .Using provinces as groups and islands/archipelagoes as populations, AMOVA revealed highly significant genetic structuring at all levels: between provinces , among islands within provinces, and within islands Table . The gen3.5FST and Jost's D differentiation tests, as well as exact Fisher's tests, were significant (after FDR correction). Pairwise FST values indicated high differentiation among all sites, ranging from 0.035*** to 0.645*** (mean ± SE = 0.348 ± 0.008), as did Jost's D values , with a maximum of 0.111*** between CHE3 and CHE8. Overall, the differentiation among sites from Grande Terre or among those from the Loyalty Islands was approximately half that of the differentiation that existed among the Western Indian Ocean sites: Within Grande Terre, values ranged from 0.064*** between GDT1 and GDT2 to 0.558*** between GDT4 and GDT9 (mean ± SE = 0.225 ± 0.020); and within the Loyalty Islands, from 0.095*** between LOY1 and LOY2 to 0.340*** between LOY3 and LOY6 (mean ± SE = 0.227 ± 0.020).All pairwise es Table . On averFST values and the ln of the geographic distances among sites , but not within islands of the Western Indian Ocean  or in New Caledonia .Mantel tests revealed a significant positive correlation between transformed 4Macrorhynchia phoenicea α . However, this result could be explained by the presence of null alleles which may occur due to some mutations in the flanking regions of microsatellite loci  intuitively enhance self‐recruitment and minimize emigration out of settled populations: larvae that settle quickly should remain close to the mother individual if they encounter suitable environmental conditions, thus forming patches of related individuals over several generations. This assumption, however, has not been tested yet.M. phoenicea α appears to reproduce throughout the year (BP pers. obs.), similarly to the tropical aglaopheniid hydrozoan Lytocarpia brevirostris  and cohort studies are necessary to resolve these issues, but this is particularly difficult in hydrozoans due to their relative small size and numerous hidden stages  different cohorts at each site or (ii) different breeding units among sampling sites, as proposed to explain the high heterozygosity deficiencies in Caribbean sponges , pointing toward extremely low gene flow at all spatial scales (supposing the same effective population size). FST values among sites were somewhat higher but comparable to those measured between L. brevirostris α populations , despite similar distances between both pairs (ca. 30 km). Along contiguous reefs, such as along the West coast of Grande Terre, FST values were comparatively low over large distances, such as between GDT3 and GDT5 . This result may be explained by a higher probability of propagules to disperse to adjacent populations along the reef over multiple generations . However, while being a useful metric, inferring causal relationships between FST and dispersal must be made cautiously as FST values can be modified by multiple processes as selection, inbreeding, drift and spatial subdivision: models linking FST to dispersal are frequently violated in natural conditions . For both aglaopheniid species, these high levels of genetic differentiation among populations might either reflect differences in current selective pressures or testify of past variations of effective population size, for example, bottlenecks or founder events, due to past climatic and geological events . Our results are congruent with findings in several other marine species, ranging from kelps to teleost fishes, for which a high pairwise differentiation was measured when habitat patches were isolated  and between coral reefs separated by expanses of open ocean: the Mantel tests indicated that geographic distances explained almost 70% of the genetic variance detected in the whole sampling. The absence of IBD at the island scale may reflect a bias in the method measuring geographic distances rather than an absence of correlation between genetic and geographic distance at such scale. Indeed, IBD was detected in the Chesterfield Islands among nearby populations (>100 km), but not in Grande Terre in spite of populations being sometimes separated by >300 km both on the West and the East coasts of the island Table . We usedM. phoenicea α are related to its life history traits. Similar to many other hydrozoans . The simplest explanation for the observed genetic structuring and low connectivity is larviparity: limited planktonic dispersal capacity induces small effective population size by reducing gene flow between populations, accelerating genetic drift. This reproductive strategy combined with the inferred capacity to successfully disperse through rafting can account for their apparent extended distribution but these traits also enhance speciation opportunities. From an evolutionary point of view, each island hosts a species (sensu Samadi & Barberousse, Our study revealed that et al., et al., The observed geographic structuring does not correspond to defined biogeographic ecoregions (Spalding et al., None declared.10.5061/dryad.cb0b8.Microsatellite genotypes are available on Dryad DOI: B.P., J.H.B., and H.M. designed the research. B.P., P.G., M.P., and H.M. generated the data. B.P., P.G., and H.M. analyzed and interpreted the data. B.P., P.G., J.H.B., M.P., and H.M. wrote the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Pilosocereus aureispinus. This species is only found in a small area of c. 300 km2 of rocky savanna from eastern Brazil and it is currently classified as vulnerable (VU) on the International Union for Conservation of Nature (IUCN) red list. Eight microsatellite loci were genotyped for 91 individuals from four localities of the known P. aureispinus range. In contrast with expectations for narrow endemic species, we found relatively high levels of genetic diversity  and very low population structure based on the variation of six loci. All the analyzed individuals were clustered in one unique genetic group in assignment tests. We also generated the sequences of two plastid markers (trnT-trnL and psbD-trnT) and found no variation on a subsample of 39 individuals. We used Landsat 8 images and Normalized Difference Vegetation Index to estimate a potential extent of occurrence of c. 750 km2 for this species. Our results showed that P. aureispinus is not suffering from erosion of nuclear genetic variability due to its narrow distribution. However, we advocate that because of the extremely limited extent of occurrence, the ongoing anthropogenic disturbances in its habitat, and phylogenetic distinctiveness of P. aureispinus, this species should be classified as endangered (EN) on the IUCN Red List.Succulent cacti (Cactaceae) are among the most threatened taxonomic groups assessed to date. Here we evaluated the genetic diversity and population structure of a narrow endemic columnar cactus  Pilosocereus  is one of the most diverse cactus genera of Eastern Brazil (44 species subdivided into two subgenera). This genus includes many species patchily distributed along mountain ranges or highlands within the Cerrado biome in South America  to select studies investigating plant species associated to the same habitats of P. aureispinus. Wilcoxon two-sample tests were performed on the by-locus (when available) or by-sample values of number of alleles per locus (A) and expected (HE) heterozygosity.Micro-Checker 2.2.3  was usedALEX 6.5 . The seqALEX 6.5  was applFST fixation index, calculated on the estimator θ [G”ST [FST values from corrected genotype frequencies to check possible overestimation of genetic differentiation induced by the presence of null alleles. We used ARLEQUIN 3.0 [FST matrix as genetic distance. The robustness of each inferred barrier was assessed by bootstrapping over loci using 100 simulated matrices of genetic differentiation. To test for evidences of population reduction, we used the heterozygosity-excess test implemented in BOTTLENECK 1.2.02 software [Level of population differentiation was assessed by using the imator θ , and by  θ [G”ST  as imple θ [G”ST . The sof θ [G”ST  was usedQUIN 3.0  to compuQUIN 3.0  was usedQUIN 3.0  as impleQUIN 3.0 , by the QUIN 3.0 , and absQUIN 3.0 . After aQUIN 3.0  was usedsoftware .FST, as suggested [The program GENECLASS2  was useduggested .P. aureispinus (http://geocat.kew.org) software [2 cells). We also estimated the potential EOO of P. aurisetus based on the association of this species with open habitats, especially those containing exposed rocks [https://glovis.usgs.gov/next/) and calculated the Normalized Difference Vegetation Index  and Pmac146 (31%) loci, which showed significant linkage disequilibrium after the Bonferroni sequential correction. Although the FST values for these two loci did not show significant difference when estimated from the original and corrected allele frequencies, as employed in FreeNA program, the inbreeding coefficient (FIS) in these loci was very different from others, causing a significant increase in mean FIS values for the analysed samples. Because of these outlier results for the Pmac135 and Pmac146 we choose to remove these loci from the downstream analyses to avoid any important distortions in recovering the genetic diversity and population structure.The program Micro-Checker recovered null alleles for all localities in A) ranged from 3.8 (IBO1) to 4.5 (IBO2); number of effective alleles (nE) ranged from 2.2 (IBO1) to 2.7 (OLB1). The observed mean heterozygosity (HO) ranged from 0.394 in IBO1 to 0.572 in OLB2, while the expected mean heterozygosity (HE) ranged from 0.390 in IBO1 to 0.525 in OLB1  for genetic diversity comparisons  to 0.095 (Pmac149) per locus. Similarly, the G"ST index resulted in an average value of 0.172 ranged from 0.061 (Pmac82) to 0.295 (Pmac128)  .According to STRUCTURE approach, a single genetic cluster (K = 1) is the most likely hypothesis to explain the distribution of genetic variation in our samples. The validity of the K = 1 hypothesis was evaluated through the Ln P(D) values of the different K values tested  and by t(IBO1) = 1.00, P(IBO2) = 0.81, P(OLB1) = 0.04, P(OLB2) = 0.56; Wilcoxon test), indicating that the populations did not undergo a recent bottleneck.The RDA analysis and Mantel test  and AOO (20 Km2 using 4km2 grid cells). Our strategy to estimate the potential EOO from the NDVI layer resulted in a larger area (app. 750 Km2) than obtained with GeoCAT. However, both estimates are within the thresholds for Endangered species . Moreover, the species has been found in just five different localities, in which two of them (OLB1 and OLB2) are only 5 km apart.The results obtained in GeoCAT assessed the species as endangered (EN). According to this analysis, the species shows extremely limited EOO  and with the congeneric P. parvus growing in campos rupestres of eastern Brazil. This picture changes when comparisons are made with both cactus and non-cactus species restricted to campo rupestre landscapes. Our results showed that the genetic diversity in P. aureispinus was lower in comparison with only two campo rupestre species, Comanthera elegans and Vellozia gigantea. In contrast, our genetic diversity estimates were comparable to three other non-cactus species and to five cactus species from genus Pilosocereus, including species as P. machrisii and P. aurisetus and the Melastomataceae Tibouchina papyrus with wide-range distributions. Taken together, these results suggest that P. aureispinus is not experiencing genetic erosion due to its restricted range; the level of genetic diversity is instead likely related to common characteristics affecting the genetic diversity both in Pilosocereus and campo rupestre species. For instance, campo rupestre landscapes has been recently proposed as an old climatically-buffered infertile landscape . The OC  did not show any variation. Besides recent selective sweep, a possible explanation to the lack of variation on the plastid markers could be a recent bottleneck of P. aureispinus. However, the moderate variation in nuclear markers and the absence of significant excess of heterozygotes prevent this later possibility. An alternative explanation could be a long-term effect of genetic drift eroding variation mostly in plastid genome, as cytoplasmic genomes in general presents fourfold smaller effective population size than nuclear genomes. Although previous studies have found intraspecific variation on cactus species in the plastid markers used in this study [Pilosocereus species [P. aureispinus could exhibit some level of variation.In contrast to microsatellite variation found in is study , includi species , lineage species . These fPilosocereus aureispinus only occurs in patches of rocky outcrops in a rugged topographic region, which may restrict connectivity in this species. Even with this patchy distribution, all analyses (except BARRIER) found no clear population genetic structure in our data. The results obtained after excluding the potentially biased loci (Pmac135 and Pmac146 loci) indicated low genetic differentiation among populations, suggesting high level of recent gene flow according to the lower FST and G"ST and small number of low-frequency private alleles. Further, STRUCTURE, DAPC, and AMOVA results suggested that P. aureispinus is composed of a single population group. In contrast, BARRIER was able to infer barriers to gene flow isolating each sampling site. Because overall and pairwise FST were low and taking into account the previous results of our population structure analyses, we conclude that BARRIER has identified a subtle, more sensible restriction to gene flow among populations. Further the analysis implemented in GENECLASS2 detected four possible migrants in 91 individuals  explanation to the lack of marked genetic structuring in P. aureispinus are the past events of expansion during glacial times that Pilosocereus species have experienced [ividuals , indicates . According to the current known distribution both methods used here suggested that the species should be raised to the Endangered (EN) category. This change of status is in consonance with the prominent phylogenetic distinctiveness of subgenus , 16. A vcurrence . This laereifera , a speciSpecies distribution ranges are usually addressed as one of the main factors to explain the level of genetic variability found in natural populations. Species with a wide and continuous distribution are supposed to have higher genetic diversity than those with restricted or endemic distribution . HoweverP. aureispinus, the genetic diversity found for this species is comparable with widespread and closely allied species [P. aureispinus present low genetic structure. The moderate genetic diversity found in P. aureispinus indicates that pollination and/or seed dispersal are efficient and promote gene flow between the localities, increasing the general effective size and reducing the impact of genetic drift. Further we concluded that this species is not suffering from deleterious genetic effects expected for species with restricted distribution, at least on the nuclear genome. Although the species has moderate to high genetic diversity, considering the extremely narrow inferred and known distribution range and its phylogenetic distinctiveness [P. aureispinus should be considered an endangered species. In order to protect against loss of genetic variability and extinction of this species, seeds are needed to be collected from as many individuals as possible and stored in seed banks. Moreover, P. aureispinus needs to be federally listed as an endangered species.To the best of our knowledge, this is the first population genetic study on a species growing in arenitic rock outcrops from Brazilian Cerrado/Caatinga boarder. In contrast with our expectation of low genetic diversity in  species , at leastiveness  we advocS1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 Information(R)Click here for additional data file.S2 Information(R)Click here for additional data file.S3 Information(DOCX)Click here for additional data file.


Paracentrotus lividus, focusing mainly on the Adriatic–Ionian basins . We applied a multidisciplinary approach integrating population genomics, based on 1,122 single nucleotide polymorphisms (SNPs) obtained from 2b‐RAD in 275 samples, with Lagrangian simulations performed with a biophysical model of larval dispersal. We detected genetic homogeneity among eight population samples collected in the focal Adriatic–Ionian area, whereas weak but significant differentiation was found with respect to two samples from the Western Mediterranean (France and Tunisia). This result was not affected by the few putative outlier loci identified in our dataset. Lagrangian simulations found a significant potential for larval exchange among the eight Adriatic–Ionian locations, supporting the hypothesis of connectivity of P. lividus populations in this area. A peculiar pattern emerged from the comparison of our results with those obtained from published P. lividus cytochrome b (cytb) sequences, the latter revealing genetic differentiation in the same geographic area despite a smaller sample size and a lower power to detect differences. The comparison with studies conducted using nuclear markers on other species with similar pelagic larval durations in the same Adriatic–Ionian locations indicates species‐specific differences in genetic connectivity patterns and warns against generalizing single‐species results to the entire community of rocky shore habitats.Connectivity between populations influences both their dynamics and the genetic structuring of species. In this study, we explored connectivity patterns of a marine species with long‐distance dispersal, the edible common sea urchin  In several cases, strong genetic partitioning was found despite a long PLD, indicating that marine populations can be less connected than expected. Limited connectivity between populations, even at small spatial scales  to investigate connectivity patterns of different species and to understand the scale at which marine protected areas (MPAs) can work as an effective network. We combined a population genomics approach with Lagrangian simulations produced by a biophysical model coupling oceanographic reanalyses (used as drivers for the movement of the planktonic stage) with information on life‐history traits affecting species dispersal. A set of genome‐wide single nucleotide polymorphisms (SNPs) was obtained using 2b‐RAD  evaluate the presence of large‐scale genetic structuring by comparing eight population samples from the Central Mediterranean (Adriatic and Ionian seas) with two population samples from the Western Mediterranean ; (2) investigate the presence of genetic differentiation at a smaller scale by comparing samples within and between the Adriatic and Ionian basins; (3) estimate potential larval connectivity and retention via Lagrangian simulations based on a biophysical model developed for the Adriatic and Ionian basins; (4) compare the patterns obtained in (3) with the realized connectivity estimated via genetic analyses, in order to provide integrated and reliable data concerning the dispersal scale of P. lividus; (5) obtain useful information for the planning and management of MPAs across this geographic area by comparison with other target species.In this context, thanks to their high frequency and genome‐wide coverage, SNP markers are expected to provide a more reliable and detailed picture of the genetic differentiation of 22.1N = 119). To this end, amplification and sequencing primers from Maltagliati et al. , 250 μmol/L of each dNTPs, 1× Reaction Buffer (RBC Bioscience), 0.05 U/μl of Taq DNA polymerase (RBC Bioscience), 20 ng of DNA, and an amplification profile consisting of six touchdown cycles of 60 s at 94°C, 30 s at 62°C with a decrease of 0.7°C per cycle and 120 s at 72°C, followed by 24 cycles of 60 s at 94°C, 30 s at 58°C, and 120 s at 72°C, and by 5 min at 72°C for final extension. PCR products were purified using QIAquick PCR Purification Kit (QIAGEN) following the instructions of the supplier and sequenced by an external service (http://www.bmr-genomics.it/). Sequences were compared to NCBI database (blastn) to confirm identification of samples as Paracentrotus lividus.A total of 275 individuals were collected at eight sampling locations in the Central Mediterranean  was cleaved with 2 U of the type 2b restriction endonucleases CspCI (New England BioLabs) overnight at 37°C, producing a population of fragments of uniform length (35 bp) with protruding ends . Amplification PCR consisted of 10 μl of ligated DNA, 0.2 μmol/L of each primer 2b‐RAD amp, 0.5 μmol/L of each primer F and barcoded primer R, 0.3 mmol/L dNTPs, 1× Phusion HF buffer, and 0.02 U/μl Taq Phusion high‐fidelity polymerase (New England BioLabs) in a total volume of 50 μl. Amplification products were run on 1.8% agarose gel stained with GelRed™ to check the quality of reactions. PCR products (1–10 μl) of different individuals were pooled according to the concentration of the target band . The amount of the PCR product from each individual was estimated by combining the concentration values obtained from NanoDrop UV–Vis spectrophotometer and the target band intensity obtained from agarose gel using ImageJ  or Genomix4Life S.r.l. , which also performed data demultiplexing and quality filtering. Each pool was sequenced twice: following sequencing results of the first run, the relative amount of the target 170 bp band of each individual was precisely estimated based on the number of reads obtained for each sample. A new pool was then assembled for each group of individuals, adjusting volumes to obtain the final normalization. The new pool was purified and sequenced as before, and the reads of each individual from the first and second Illumina sequencing run were merged and analyzed together. This procedure allowed for an increase in the depth coverage and ensured obtaining an equal and comparable number of reads from each analyzed sample.2.4http://www.bioinformatics.babraham.ac.uk/projects/fastqc/); then, custom‐made scripts were used to filter reads for the presence of CspCI recognition sites and to trim adaptors, obtaining sequences of uniform length (32 bp). The trimmed, high‐quality reads  were used for subsequent analysis. In silico assembly of loci and genotyping was performed using STACKS software  of 15; number of mismatches allowed between stacks to build a locus in an individual (M) equal to 4; maximum distance between loci from distinct individuals to be merged in the population catalog (n) equal to 4; error rate to call SNP (bound) between 0 and 0.1; significance level required to call a heterozygote or homozygote  equal to 0.05. The STACKS module Populations was used to generate output in GENEPOP format for further downstream analysis. As the minimum coverage for each allele per locus was set to 15 , no further filtering by sequencing depth was applied. CREATE software  and used to create a local database. Consensus sequences of a subset of 2b‐RAD loci with different polymorphism levels were used as query for a local blastn against the EST‐linked sequences database. Consensus sequences of each locus and its matching EST‐linked sequences were aligned using CLUSTALW Omega tool (available online at http://www.ebi.ac.uk). Primers for Sanger sequencing were designed on a selection of 10 loci using sequence information of alignments and tested on a small number of individuals with a known genotype. PCR conditions were optimized depending on annealing temperature of each pair of primers and on the expected amplicon length  and unbiased expected heterozygosity (He) with GENETIX 4.05.2  was calculated using HP‐RARE  across all population samples.The genetic variability within population samples was assessed on polymorphic loci by computing the observed heterozygosity (FST), as well as nonhierarchical and hierarchical analysis of molecular variance  were tested. A wide range of population sizes was used , including the smallest effective population size (Ne) value estimated from P. lividus microsatellites temporal variation  of 0.01, and the infinite allele model; for each run, three replicates were performed. BAYESCAN was run with burn in = 50,000, thinning interval = 30, sample size = 5,000, number of pilot runs = 50, length of pilot runs = 5,000, and the same false discovery rate (FDR) threshold set in LOSITAN (0.01). In both cases, all the polymorphic loci were used and the comparisons were performed between Adriatic–Ionian population samples and the two additional population samples from the Western Mediterranean Sea, as well as among Adriatic–Ionian population samples. The allele sequences of all loci identified as putative outliers were searched against a P. lividus transcriptome database of 188,000 contigs .Potential connectivity of http://oceanlab.cmcc.it/afs). Daily average fields of current velocity and temperature cover the whole Adriatic basin down to the 39°N parallel in the Ionian Sea over a regular horizontal grid with a resolution of 1/45° (about 2.2 km) and 31 vertical sigma layers. Bathymetry has a horizontal resolution of 1/60°, and the coastline is set in correspondence with the 10‐m isobath.The oceanographic engine of the biophysical model is based on the ocean circulation dataset produced by the AREG model  and 9 years  reported in Penant et al. . After trimming and filtering for restriction site, a total of 992,809,025 reads of 32‐bp length were retained (92%), resulting in about 3.5 million reads per individual. An average of 25,000 tags per individual was obtained, leading to an approximate coverage per locus of 140. In general, a uniform read coverage between samples for the same locus was observed, whereas variability was observed between different loci. A total of 212,260 2b‐RAD tags were identified among 276 individuals, 138,745 (65%) were monomorphic and 73,515 (35%) were polymorphic. After filtering, we retained 1,122 polymorphic loci for further analysis. The technical replicates matched at 1,112 of 1,122 loci; nine of ten differences were due to missing loci between the two replicates. Of the 1,122 loci, 261 loci had only one SNP, 420 had two SNPs, and 441 had three SNPs, for a total of 2,424 SNPs. As mentioned in Section P. lividus ; considering that PCR primers were designed on EST‐linked sequences, this result probably reflects the presence of introns within these PCR products. The remaining four loci were successfully sequenced, and the 2b‐RAD genotypes were confirmed Table .3.4p > .05).Comparable values were obtained among all the population samples for observed heterozygosity, unbiased expected heterozygosity, and allelic richness Table , with on3.4.1FST was highly significant (p < .0001), and its 95% confidence interval (CI) did not include 0 (0.00435–0.00828), indicating the presence of genetic structuring across the study area. All sixteen pairwise FST comparisons between Adriatic–Ionian and the Western Mediterranean population samples were highly significant, with FST values ranging from 0.00663 to 0.02475  was also highly significant after correction for multitest. Hierarchical AMOVA showed that the genetic variation can be partitioned into three geographic groups, the first including the eight Adriatic–Ionian population samples, the second including only the population sample from France, and the third one including the sample from Tunisia ; also in this case, the 95% CI did not include 0 (0.0089–0.01823). This subdivision into three different geographic pools maximized the amount of variation among groups with respect to any alternative configuration including either two or three groups. Notably, the subdivision into two groups as proposed in the literature . This genetic pattern was confirmed by DAPC  and Western Mediterranean (France and Tunisia), the analysis of molecular variance showed that most of the variation (>99%) occurred within populations and only 0.63% among populations. However, the corresponding global FST values among the eight Adriatic–Ionian population samples were all negligible (FST ranging from −0.00075 to 0.00559) and statistically not significant after multiple test correction .At the basin scale, by contrast, pairwise FST of Adriatic–Ionian population samples can be reached and exceeded in a few generations (Table FST (0.00217). This maximum value was obtained using a population size of 10,000, whereas considering a value of 100  when comparing Central and Western Mediterranean population samples . Due to some redundancy in P. lividus transcriptome, in six of the nine cases . Similarly, the hierarchical AMOVA on the three groups identified above  confirmed the genetic structure obtained with the full dataset . The pairwise FST between Central and Western Mediterranean samples and between France and Tunisia remained small but highly significant after correction for multitest, and comparisons among Adriatic–Ionian samples were all nonsignificant.The pattern of genetic structure obtained with all 1,122 loci was confirmed also when excluding the 17 putatively selected outliers from the dataset Table . For ins3.5The strength of each sampling location to serve as a direct source of propagules for other locations is summarized through pie diagrams in Figure P. lividus in the focal area, this means that only in some cases do the propagule fluxes directly connect sampling locations that are not nearest neighbors. Particles originating from Greek coasts (OTH) were able to reach Montenegro (BOK), and particles from both OTH and KAP were successful in crossing the Adriatic Sea and reaching some Italian sampling locations (OTR and POC). All Italian locations were connected with each other from north to south (TRE to POC), in most cases through persistent (>0.75) connections. TRE acted as an important source of particles for all Italian locations, while POC acted as an effective receptor of particles coming from most locations on both sides of the basin . Some particles were occasionally able to cross the Adriatic Sea from west to east, starting from TOG and OTR.Estimates of connectivity effectiveness Table  and persp > .2), while survival showed a significant (p = .01) negative trend (y = −0.015t + 31.05), suggesting that changes in water temperature may have determined a reduction in larval survival over the last decade . POWSIM analysis , reflecting the small size of the population samples in the original study (N = 10 each). By contrast, the power of our panel of SNPs estimated by using the same number of generations and a fourfold greater diploid effective population size , was very high, approaching 100% in the majority of the cases. This result clearly indicates that the detection of differences between Adriatic–Ionian samples with cytb sequences but not with our panel of SNPs is very unlikely under the simulated conditions.The available cytb sequences for Adriatic and Ionian samples  predicted a potential larval exchange among locations, altogether supporting the connectivity of P. lividus in this area from a seascape genetics perspective.Overall, the 2b‐RAD population genomics analysis of the eight Adriatic–Ionian and two French‐Tunisian population samples of 4.1FST values between Western and Central Mediterranean samples, although highly significant, were small, indicating that shallow differentiation exists also at this very wide geographic scale .Our results strongly support evidence of genetic partitioning among population samples collected throughout the entire study area, with a clear genetic break separating Central (Adriatic–Ionian) and Western (France–Tunisia) Mediterranean samples. This result is consistent with previous studies  against any alternative partitioning including only two groups.P. lividus, and six of them have a correspondence with Strongylocentrotus purpuratus proteins in the database of NCBI. The most intriguing match is for the fibrillary glycoprotein hyalin .Whether or not the putative outliers are taken into account, the results of our molecular analysis highlight an incontrovertible genetic homogeneity among the eight Adriatic–Ionian population samples. Both the analysis of molecular variance and all pairwise comparisons strongly support a pattern of genetic homogeneity in this area, where all the analyzed population samples seem to belong to a single panmictic population, at least from the genetic point of view. None of the pairwise FST value obtained for the Adriatic–Ionian basins. Taking into account the population sizes used in forward‐time simulations  are sufficient to quickly and stably reach the ns Table , these sP. lividus are widely available across the surveyed area. It is therefore very likely that stepwise, along‐shore connections between unsampled locations can greatly increase the potential for dispersal among the modeled locations. For all these reasons, we believe that our general picture of potential dispersal, suggesting a general mixing within the Adriatic–Ionian seas, provides support for the hypothesis that the genetic homogeneity observed in the area is due to exchange between locations.Notably, a scenario of genetic homogeneity with gene flow is consistent with the potential for larval connectivity obtained via Lagrangian simulations, performed by taking into account species‐specific life traits of spawning and dispersal. Simulations suggest that each sampling location within the Adriatic–Ionian basin is potentially connected to its closest neighbor  through a relatively persistent flux of propagules. In addition to these along‐shore connections between neighboring locations, a few cross‐basin dispersal events (between the eastern and western coasts of the Adriatic) can take place in both directions and may sometimes reach also the furthest (Ionian) locations. In this regard, it is important to note that, according to our settings, the Lagrangian simulations should provide lower‐bound (conservative) estimates of potential connectivity. This conservativeness is the result of at least three major restrictions. First, we considered only a single spawning season between April and July , with significant FST values among “nearly all population pairs within the Mediterranean and Adriatic basins, some of which were geographically close (about 40–60 km between our populations from the French Mediterranean coast)” and ΦST values capturing only differences at major transitions. This pattern was explained by a lack of informativeness of mtDNA mutations at the local scale, contrasted by the possibility to obtain significant FST values after relatively few generations of genetic drift , a relatively sedentary species . Our results are consistent with the evidence of complete homogeneity (obtained via both microsatellites and Lagrangian simulations) for the marbled crab P. lividus populations should be managed as a single unit, because the amount of migration needed to maintain genetically homogeneous populations is far smaller than the amount required to make them indistinguishable from a demographic viewpoint  to the planning and management of MPA networks might be necessary. However, from a management perspective, it is essential to consider that the lack of a detectable genetic differentiation does not necessarily imply that s Tables  and 4, aScorpaena porcus), a rocky shore fish characterized by a PLD of about 1 month, microsatellites genetic analyses pointed out a clear subdivision for the same species between the eastern and western part of the Adriatic and Ionian basins  indicates that genetic homogeneity is not so common. For instance, while multispecies biophysical modeling , together with differences in the spatial distribution, abundance and fecundity of their source populations, as well as recruitment success , can indeed produce very different connectivity outcomes even when PLDs are very similar.Although still based on a relatively small number of species, this comparison clearly warns against inferring a general pattern based only on the duration of the dispersal phase of each species. While a propagule duration of about 1 month may be sufficient to ensure genetic mixing in some species, such as 5P. lividus at the largest geographic scale within the Mediterranean Sea, and (ii) genetic mixing in the Adriatic–Ionian seas, where a smaller geographic scale and/or peculiar oceanographic features allow for an efficient exchange among locations for a species with long‐distance dispersal like P. lividus. However, comparison with the few published studies regarding connectivity conducted in the same area, even in species with a similar PLD, showed the existence of different patterns of genetic differentiation, ranging from complete panmixia to an east–west or north–south genetic partitioning. These findings warn against any generalization based on the presently available information and strongly indicate the need to obtain genetic connectivity data from a wider array of species, including keystone species such as habitat formers as well as rare or threatened species. At the same time, detailed biological data are needed to refine biophysical models, such as basic information about the correlation of physical and biological drivers of reproductive timing and larval mortality, which both have a strong influence on connectivity. The integration of all these aspects represents a crucial step toward providing a general framework of connectivity at the community level, and predicting expected changes in community structure in the future. It is also a crucial step toward the holistic perspective needed to achieve the Aichi Target 11 of the Convention on Biological Diversity, which aims at increasing the proportion of protected areas in the world, working as “a well‐connected system” by 2020  genetic differentiation in 10.5061/dryad.73kc6. Short read data are deposited in SRA with accessions SRR5266202‐SRR5266474.SNPs genotypes data together with the datasets supporting the conclusions of this article are available as supporting information and additional files in DRYAD at doi:None declared.MP, PM, MS, and LZ conceived and designed the experiments and wrote the manuscript. MP and MS performed the experiments. MP, PM, MS, and LZ analyzed the data. PM, LZ, and TP contributed reagents/materials/analysis tools. GA, JBS, EB, RC, AC, MC, LC, GG, CK, VM, IAMM, PM, CP, TP, MP, MS, and LZ critically reviewed the manuscript; the final version of the manuscript was approved by all the authors. Click here for additional data file.


The USDA Agriculture Research Service National Plant Germplasm System (NPGS) preserves the largest sorghum germplasm collection in the world, which includes 7,217 accessions from the center of diversity in Ethiopia. The characterization of this exotic germplasm at a genome-wide scale will improve conservation efforts and its utilization in research and breeding programs. Therefore, we phenotyped a representative core set of 374 Ethiopian accessions at two locations for agronomic traits and characterized the genomes.FST values ranging from 0.11 to 0.47. In total, 198 accessions (53%) were assigned to one of these populations with an ancestry membership coefficient of larger than 0.60; these covered 90% of the total genomic variation. We characterized these populations based on agronomic and seed compositional traits. We performed a cluster analysis with the sorghum association panel based on 26,026 SNPs and determined that nine of the Ethiopian populations expanded the genetic diversity in the panel. Genome-wide association analysis demonstrated that these low-coverage data and the observed population structure could be employed for the genomic dissection of important phenotypes in this core set of Ethiopian sorghum germplasm.Using genotyping-by-sequencing, we identified 148,476 single-nucleotide polymorphism (SNP) markers distributed across the entire genome. Over half of the alleles were rare (frequency < 0.05). The genetic profile of each accession was unique , and the average genetic distance among accessions was 0.70. Based on population structure and cluster analyses, we separated the collection into 11 populations with pairwise The NPGS Ethiopian sorghum germplasm is a genetically and phenotypically diverse collection comprising 11 populations with high levels of admixture. Genetic associations with agronomic traits can be used to improve the screening of exotic germplasm for selection of specific populations. We detected many rare alleles, suggesting that this germplasm contains potentially useful undiscovered alleles, but their discovery and characterization will require extensive effort. The genotypic data available for these accessions provide a valuable resource for sorghum breeders and geneticists to effectively improve crops.The online version of this article (doi:10.1186/s12864-016-3475-7) contains supplementary material, which is available to authorized users. Sorghum bicolor (L.) Moench] is the fifth most important grain crop after maize, wheat, rice, and barley [Sorghum [d barley . Its rold barley , but thed barley . These rd barley , and ongEx situ sorghum germplasm collections  have been established in several countries as a resource for breeding programs. These germplasm collections provide genetic variation for crop improvement programs. Today, the largest worldwide sorghum collection is maintained by the USDA Agriculture Research Service National Plant Germplasm System (NPGS) and includes >41,860 accessions from 114 countries. Likewise, the International Crops Research Institute for the Semi-arid Tropics (ICRISAT), located in India, has a sorghum collection of 37,904 accessions from different countries. The National Genebank of China maintains a sorghum collection of over 16,000 accessions, including 12,000 Chinese sorghum landraces. To use these resources, breeding programs must have an understanding of the variation within the germplasm collection . The estThe selection of landraces for inclusion in a core set based on passport information alone may not adequately represent the genetic diversity owing to limited passport information for many accessions and/or genetic redundancy . Therefore, the use of a molecular genetics approach is imperative for the conservation and utilization of the collection. However, molecular genetic approaches have been limited to specific samples of sorghum from the NPGS , 10–13, Genome sequence information and next-generation sequencing technology have made important contributions to the development of new genotyping platforms. Today, genotyping-by-sequencing (GBS)  is arguaThe Ethiopian germplasm maintained by NPGS contains 7,217 accessions, representing 17% of the sorghum collection . UnfortuThe Ethiopian germplasm core set examined in this experiment comprised 352 exotic accessions and 22 converted tropical lines that are photoperiod-insensitive and dwarf and were produced by crossing exotic lines and modern US cultivars. This core set represented ~5% of the Ethiopian collection, including 16 sorghum races, of which Durra, Durra-bicolor, and unclassified accessions represented 72% of the accessions (Table r2 < 0.5) that were evenly distributed across the sorghum genome. Remarkably, of the 69,564 pairwise IBS genetic distances, only 14 exceeded 0.90  may contribute to problems, such as material duplication by labeling and logistic errors. We calculated the identity-by-state (IBS) genetic distance among all pairs of accessions using 27,306 unlinked SNPs (FST) among these populations ranged from 0.11 to 0.47, indicating a relatively high level of genetic differentiation (Table e) based on the linkage disequilibrium (LD) of 58,634 SNPs with MAF > 0.05. We obtained an Ne of 199 individuals, which was comparable to the total number of accessions with an ancestry membership coefficient of greater than 0.60. In fact, these 198 accessions covered 90% of the observed allelic diversity ; accordingly, additional phenotyping could be limited to or initiated with these accessions. The conservation of these particular accessions is imperative to maintain the genetic diversity of this core set of the collection.Understanding the population structure of the NPGS Ethiopian collection is imperative to establish new strategies to preserve and utilize its genetic diversity and is a prerequisite for GWAS. We inferred that there are 11 ancestral populations based on 10-fold cross-validation using ADMIXTURE  Fig. 2a. Indeed,We used an unrooted neighbor-joining tree to determine the genetic relationships among these populations. The results of this analysis were consistent with the previously determined population structure Fig. , with 11Based on the allele frequency distribution for this NPGS Ethiopia collection core, 60% of the SNPs were rare (MAF < 0.05) Fig. . A high How much of the genetic diversity present in NPGS Ethiopian germplasm has been used in US breeding programs? The US SAP encompasses a wide range of sorghum germplasm that has been exploited in breeding programs and was recently genetically characterized . We idenr2) and FST across the genome. In the Ethiopia collection, we observed an average r2 of ≤0.2 at distances of greater than 6 kb  between these 11 Ethiopian populations (Table FST outlier method implemented in BayeScan v.2.1 [Tannin1 [FST = 0.62). Tannins have diverse biological functions, including protection against pathogenic bacteria and fungi and predation by herbivorous animals [FST outlier SNPs with a broad range of gene ontology annotations, including metabolic process (glycosyl transferase), signal transduction (auxin response factor), DNA damage repair, and retrotransposon proteins. Natural selection contributes more substantially to the structure of Ethiopian sorghum populations than selection by farmers.The population structure of a plant is determined by abiotic and biotic factors that influence selection . The higns Table  indicatean v.2.1 . This ap[Tannin1   to identify the relative importance of these traits in shaping phenotype diversity. The first four principal components accounted for approximately 60.5% of the total phenotype diversity, and seed compositional traits had the largest loading coefficients , 7 (dw3), and 6 (dw2)  without including the 22 converted dwarf tropical lines. We detected a significant association between PH and the distal region of chromosome 6 , we identified two loci in the GWAS that were 57 kbp apart  and S2_57662361 (p < 1.2−6), respectively, which explained 11.5 and 9.6% of the genetic variance, respectively. Since the genetic relatedness among accessions only accounted for 23.3% (fat) and 21.1% (protein) of the genetic variance other important loci are still unidentified. These SNPs were located within the genes Sb02g023740.1 (unknown protein) and Sb02g023770.1 (cytochrome P450), which have not been associated with particular phenotypes in sorghum or Arabidopsis. Additional phenotype and genotype analyses are required to verify these associations. This Ethiopian core set and its genetic characterization could provide a basis for the discovery of important loci in this exotic germplasm.Based on a GWAS of seed compositional traits, we identified novel loci for protein and fat content, and confirmed a previous result for total phenol content. We obtained similar results for total phenol and tannin contents; SNP S4_61667908 demonstrated the most significant association for these traits  for temperate grain production regions via the introgression of dwarfing and early maturity genes . Today, bidopsis , rice [3bidopsis , and whebidopsis  identifiToday, the SAP is widely used for the genomic dissection of multiple agronomically important traits , 34, 41.Germplasm conservation is imperative for continual agricultural improvements. Studies of the genetic relatedness among all ex situ accessions in germplasm collections and genetic redundancy should improve conservation efforts and practical applications. GBS is rapidly becoming the standard choice for genetic studies and germplasm characterization. Hence, in the near future, multiple independent GBS analyses of sorghum accessions from different germplasm collections will likely be integrated into a meta-analysis to identify duplicates and closely related accessions among worldwide germplasm collections. In this regard, a multi-institutional effort is necessary to systematically explore the genetic diversity of all ex situ sorghum collections to broaden the genetic base of breeding programs worldwide. Indeed, the preservation of redundant germplasm in ex situ collections is a waste of limited resources  and doesThe NPGS Ethiopian sorghum germplasm is a highly genetically and phenotypically diverse germplasm collection comprising 11 populations. The genetic diversity of the germplasm is largely unused in sorghum breeding programs. Our results provide sorghum breeders and geneticists worldwide with knowledge and tools to utilize and conserve this germplasm. In fact, these results will facilitate resource optimization by improving experimental designs based on population structure and genetic diversity. Moreover, this genomic characterization is the first step toward the development of a NPGS sorghum core collection based on genetic profiles.The 7,237 Ethiopian sorghum accessions in the current NPGS collection were grouped according to the GRIN database into the 16 known sorghum races and one unknown category . In both locations, a completely randomized design with plots of 1.8 m in length with 0.9 m between rows was used. Plants were maintained using standard management practices, and weeds were controlled with mechanical tillage and hand hoeing.r2 = 0.81), width (r2 = 0.92), diameter (r2 = 0.78), and area  (unpublished observations). Thus, seed size referred to the average volume of 100 seed from three representative panicles per accession.FL, PH, PL and PD, the ratio of PL/PD, and seed size were obtained for each accession/plot at both locations. FL was defined as the number of days until 50% of the plants within a plot reached anthesis. PH referred to the average distance from the base of the main stalk  to the top of the panicle at maturity for two representative plants per accession/plot. PL and PD referred to the distance from the first rachis to the top of the panicle, and to the widest section of the panicle , while RA compositional analysis of whole seeds was performed using near-infrared (NIR) spectroscopy  in 2013.Early-season cold tolerance was determined using the average cold germination (G72) for fresh seeds from 2013 according to the procedures recommended by the Association of Official Seed Analysts . The linLeaf dhurrin content is a quantitative measure of the level of pre- and post-flowering drought tolerance . In 2013Y = μ + L + P + L x P + e; where Y is the trait, μ is the common effect, L is the location effect, P is the population effect, L x P is the year × location effects and e is the plot to plot variation within populations. The ANOVA for traits with one location data was the following Y = μ + P + e. The means of significant different traits (p <0.05) among Ethiopian population was compared using the Fisher’s least significance difference test.The combined data over all locations were used to standardize the 16 traits to a mean of zero and a standard deviation of one for multivariate analysis. PCA was performed using the standardized data with a covariance matrix in SAS. Likewise, analysis of variance (ANOVA) were performed using the subset of 198 accessions assigned to the 11 subpopulations. The linear effect model for such ANOVA was the following: http://www.biotech.cornell.edu/brc/genomic-diversity-facility) using the restriction enzyme ApeKI for digestion. The library had 384 unique barcodes including 4 random blanks . The GBS library was sequenced on four lanes (depth = 5.0) of an Illumina HiSeq 2500, and the GBS analysis pipeline (Tassel Version: 3.0.147), an extension of the Java program TASSEL [Sorghum bicolor v.1.0; [Genomic DNA was isolated from seedlings of the 374 Ethiopian accessions based on the methods of Guillemaut, Marechal-Drouard . DNA wasr v.1.0; ). To ensr v.1.0; .r2 < 0.5) distributed across the genome were identified with PLINK [Population structure and admixed ancestry were assessed using a model-based clustering method implemented in ADMIXTURE . Twenty-th PLINK  and usedth PLINK .R2 between SNPs with a p-value of <0.001. Effective population size was determined based on LD values from SNPs with a minor allele frequency of >0.05 as implemented in SNeP [LD between SNPs for each chromosome was estimated in TASSEL 5.0  using a  in SNeP .FST outliers were detected among the 27,306 unlinked SNPs using BayeScan v.2.1 [FST outliers was determined using a false discovery rate of 0.001 as implemented in the “plot_bayescan” function in R.The pairwise fixation index among populations was estimated based on the method of Weir and Cockerham  using than v.2.1 . To reduPairwise genetic distances among the 374 Ethiopian accessions were calculated based on IBS as implemented in TASSEL 5.0  using 27r2 < 0.5) SNPs were identified in PLINK [Genetic relationships among the NPGS Ethiopia collection and SAP were investigated by the identification of common SNPs in the SAP  and NPGin PLINK  for furtin PLINK , and thein PLINK .p-values were examined to determine how well the models accounted for population structure and familial relatedness. The phenotypic data of PD, RA, and tannin content were normalized prior to the association analysis. Significant associations were determined for each trait using a false discovery rate-adjusted p-value of >0.05 [The presence of population structure and familial relationships among accessions must be adequately accounted for in the GWAS model approach to reduce spurious associations . The assof >0.05  as impleof >0.05 .


Fusarium graminearum complex are the main pathogens associated with the disease, F. graminearumsensu stricto being the main pathogen in Argentina. Biocontrol can be used as part of an integrated pest management strategy. Phytohormones play a key role in the plant defense system and their production can be induced by antagonistic microorganisms. The aims of this study were to evaluate the effect of the inoculation of Bacillus velezensis RC 218, F. graminearum and their co-inoculation on the production of salicylic acid (SA) and jasmonic acid (JA) in wheat spikes at different periods of time under greenhouse conditions, and to evaluate the effect of B. velezensis RC 218 and Streptomyces albidoflavus RC 87B on FHB disease incidence, severity and deoxynivalenol accumulation on Triticum turgidum L. var. durum under field conditions. Under greenhouse conditions the production of JA was induced after F. graminearum inoculation at 48 and 72 h, but JA levels were reduced in the co-inoculated treatments. No differences in JA or SA levels were observed between the B. velezensis treatment and the water control. In the spikes inoculated with F. graminearum, SA production was induced early (12 h), as it was shown for initial FHB basal resistance, while JA was induced at a later stage (48 h), revealing different defense strategies at different stages of infection by the hemibiotrophic pathogen F. graminearum. Both B. velezensis RC 218 and S. albidoflavus RC 87B effectively reduced FHB incidence (up to 30%), severity (up to 25%) and deoxynivalenol accumulation (up to 51%) on durum wheat under field conditions.Fusarium head blight (FHB) is a devastating disease that causes extensive yield and quality losses to wheat and other small cereal grains worldwide. Species within the  Triticum aestivum L.) and durum wheat (Triticum turgidum L. var. durum) can be affected by Fusarium head blight (FHB). Within the Fusarium graminearum species complex, Fusarium graminearum sensu stricto is the main pathogen associated with FHB in Argentina. During the last 60 years, several epidemics of FHB of varying degrees of severity have occurred in Argentina. During 2012, a severe FHB outbreak occurred in the main wheat growing area, with estimated losses of up to 70% -SA and 50 ng [2H6]-JA. Centrifugation was performed at 5000 rpm for 15 min, the pellet was discarded, the pH of the supernatant was adjusted to 2.8 with acetic acid, and the supernatant was partitioned twice against an equal volume of diethyl ether . The aque et al. . Solvent®; Advion BioSciences, Ithaca, NY, USA). For stable nanoESI, 70 µL min−1 of 2-propanol/acetonitrile/water  containing 0.3 mmol/L NH4HCOO (adjusted to pH 3.5 with formic acid) delivered by a Pharmacia 2248 HPLC pump  were added just after the column via a mixing tee valve. By using another post column splitter, 502 nL min−1 of the eluent were directed to the nanoESI chip with 5 µm internal diameter nozzles. The ionization voltage was set to −1.7 kV. Phytohormones were ionized in a negative mode and determined in a scheduled multiple reaction monitoring mode with an AB Sciex 4000 QTRAP® tandem mass spectrometer . Mass transitions were as follows: 215/59  −35 V, entrance potential (EP) −8.5 V, collision energy (CE) −24 V) for D6-JA, 209/59  for JA, 141/97  for D4-SA and 137/93  for SA. The mass analyzers were adjusted to a resolution of 0.7 amu full width at half-height. The ion source temperature was 40 °C, and the curtain gas was set at 10 (given in arbitrary units) . Fusarium-damaged kernels (FDK) is another common parameter used as an indicator of disease severity. A total of 300 grains per treatment were analyzed and FDK was defined as the percentage of scabby grains  present in the sample.p ≤ 0.05). FHB disease severity were subjected to ANOVA on ranks and means were separated by Dunn’s method (p ≤ 0.001). Deoxynivalenol accumulation data were subjected to a one way ANOVA and means were separated by Duncan’s method (p ≤ 0.05). DNA quantification and FDK data were subjected to a one way ANOVA and means were separated by the Fisher and Holm–Sidak methods, respectively (p ≤ 0.05). All statistical analyses were performed using SigmaStat for Windows version 3.5 .Phytohormone’s data were subjected to one way analysis of variance (ANOVA) and means were separated by Tukey’s method (