Brucella ovis causes an infectious disease responsible for infertility and subsequent economic losses in sheep production. The standard serological test to detect B. ovis infection in rams is the complement fixation test (CFT), which has imperfect sensitivity and specificity in addition to technical drawbacks. Other available tests include the indirect enzyme-linked immunosorbent assays (I-ELISA) but no I-ELISA kit has been fully evaluated.B. ovis.The study aimed to compare an I-ELISA kit and the standard CFT. Our study was carried out on serum samples from 4599 rams from the South of France where the disease is enzootic. A Bayesian approach was used to estimate tests characteristics . The tests were then studied together in order to optimise testing strategies to detect I-ELISA = 0.917 , 95% Credibility Interval (CrI) compared to Se CFT = 0.860 , 95% CrI). However, CFT was slightly more specific than I-ELISA  compared to Sp I-ELISA =0.952 , 95% CrI).After optimising the cut-off values in order to avoid doubtful results without deteriorating the concordance between the results of the two tests, the I-ELISA appeared to be slightly more sensitive than CFT (Se The tests were then associated with two different interpretation schemes. The series association increased the specificity of screening and could be used for pre-movement testing in rams from uninfected flocks. The parallel association increased sequence sensitivity, thus appearing more suitable for eradicating the disease in infected flocks.The high sensitivity and acceptable specificity of this I-ELISA kit support its potential interest to avoid the limitations of CFT. The two tests could also be used together or combined with other diagnostic methods such as semen culture to improve the testing strategy. The choice of test sequence and interpretation criteria depends on the epidemiological context, screening objectives and the financial and practical constraints. Brucella ovis is a Gram-negative coccobacillus. In sheep, B. ovis infection is responsible for a reproductive disease, often causing genital lesions such as unilateral or bilateral epididymitis in rams and, more rarely, abortion in ewes. This disease mainly spreads via venereal transmission, even though other routes of infection have been observed. Infected ewes generally clear the micro-organism from the vagina within two oestrus cycles )Our study compared a commercial I-ELISA kit and standard CFT in order to evaluate its potential use as a complementary test for international trade or for diagnosis on farms, and to assess its possible use instead of CFT or in association with it to improve the detection and eradication of the disease and the protection of disease-free flocks.B. ovis infection is enzootic: 3,063 were from Pyrénées Atlantiques . A free population used as a source of prior information on the specificity of I-ELISA included 3,792 rams selected from free artificial insemination (AI) units.The study included sera from 4,599 rams collected in the South of France, in areas where Blood samples were collected as part of the usual official screening scheme on farms  The EU recommendations about screening procedures and EU ethical guidelines and animal welfare regulations were strictly respected.B. ovis, Idexx, France) were performed in parallel on all sera from the infected populations described above. The antigens used for both CFT and I-ELISA consisted in a hot-saline water soluble extract of the REO 198 strain prepared according to OIE recommendations )Brucella-R-LPS antibodies. They are consequently expected to be conditionally dependent )termined . Table 1Prior information on CFT combined literature and experts’ advice with field data. Only prior information from similar studies in literature was input into the model. According to previous studies, mean CFT sensitivity ranged from 88.9% to 98.7% and mean CFT specificity from 89.3% to 100%. The lowest sensitivity reported was 77% and the lowest specificity 78% ,29-33).-33.29-33Prior information on I-ELISA specificity (Sp I-ELISA) was input from a study performed on the sera from the 3,792 rams selected in officially brucellosis free AI units. As these rams were being raised in specific conditions, with little contact with environmental pathogens potentially responsible for cross-reactions, they were not representative of the population bred on farms in the South of France. They were not therefore studied with the population of 4,599 rams from the South of France but used to provide prior information on the specificity of I-ELISA (Sp I-ELISA = 99.74%).Since little information was available in the literature on this I-ELISA sensitivity and since the corresponding studies were carried out with very different methods, in different contexts, as indicated above, beta  uninformative distributions (uniform distributions between 0 and 1) were chosen.Prevalence in the population and covariance of the sensitivity and the specificity were unknown: beta  uninformative distributions were also used as priors for these parameters.The statistical analysis was performed with the WinBUGS program . The MCMSERIES = SeIELISA*SeCFT + γSe and its specificity was: SpSERIES = (1 - SpIELISA)*(1 – SpCFT) – γSp. The sensitivity of the association in parallel was: SePARALLEL = 1-((1-SeIELISA)*(1-SeCFT)) – γ Se and its specificity was: SpPARALLEL = SpIELISA * SpCFT + γ Sp; .After having estimated the sensitivity and the specificity of the two tests, their characteristics were evaluated when used in association . With thA new cut-off was established for I-ELISA in order to eliminate doubtful results without affecting the concordance between the results of CFT  and I-ELISA. A cut-off at 45% OD (see above) was the best compromise to optimise both the concordance of positive results and the concordance of negative results. With this new cut-off, the two tests gave concordant results (positive / positive or negative / negative) for 4,161 rams (92.6%).The apparent prevalence assessed in the whole studied population by the I-ELISA kit was 25.6%. In the subpopulations 1, 2 and 3, the apparent prevalences were respectively 17.0%, 44.8% and 27.6%. Table The I-ELISA kit was slightly more sensitive than the CFT  whereas Se CFT = 0.860 , 95% CrI). In contrast, the CFT was somewhat more specific than the I-ELISA .-7; 0.0361], 95% CrI).Prevalence in the studied population of 4,599 from the South of France was evaluated at 0.238 . The sensitivity covariance was 0.0241  and the specificity covariance was 0.00521  and its specificity was SpSERIES = 0.995  (95% CrI). The diagnostic sensitivity of the parallel association was SePARALLEL = 0.964  and its specificity was SpPARALLEL = 0.946 .The diagnostic sensitivity of the association in series was SeB. ovis infection is enzootic )i.e. showing only negative results to regular tests), so the positive predictive value of the test results is low. For this reason, a high cut-off value could be used (for instance 60% OD). In contrast, when the test is used to diagnose B. ovis infection in infected areas or to confirm the infection in rams with clinical lesions with the aim of eradicating the disease, false negative results might slow down eradication and, in particular conditions, it could be acceptable to cull seropositive but uninfected rams. Accordingly, in a context of high prevalence, a low cut-off value could therefore be used (for example 30% OD).In a screening situation, one important issue is the interpretation of doubtful results obtained with the I-ELISA. We established a cut-off different from that recommended by the manufacturer to avoid doubtful results without decreasing the concordance between the results of the two tests. Nevertheless, it is possible to adjust this cut-off according to the epidemiological context. When the test is used for international or intra-community trade, false positive results due to a lack of specificity could hinder trade and raise economic issues in brucellosis free contexts. Exported rams should come exclusively from uninfected farms  to confirm or rule out the infection.B. ovis has already been identified, the testing regime should be focused on a quickly eradication of the disease. In this case, all animals  should be submitted to I-ELISA and CFT in parallel. Animals with positive results to at least one of the two tests should be culled and replaced by virgin animals from flocks free of B. ovis infection. The remaining animals resulting negative to both tests could be kept in the flock but should neither be sold nor exported. Further tests should be carried out at 45- to 60-days intervals until all infected animals have been eliminated . This strategy raises financial issues in infected flocks with a high prevalence of the disease, because many animals would be culled.In infected flocks in which Brucella ovis.In all flocks, animals should be submitted to a clinical examination two months before the breeding season. Rams with clinical signs of epididymitis should be banned from reproduction and subjected to an I-ELISA test. Rams with positive results to I-ELISA should undergo further investigations (semen culture) to confirm the presence of B. ovis infection in sheep. We showed that this I-ELISA kit was slightly more sensitive than the CFT, but somewhat less specific. The high sensitivity and acceptable specificity of the I-ELISA kit support its potential interest for diagnosing B. ovis infection in sheep. This I-ELISA kit has certain advantages over CFT and could be used alone or as a first test in associated testing regimes. The interpretation of the results and the testing sequences should be chosen depending on screening objectives, the disease’s prevalence in the target population and economic and practical constraints.Our study used a Bayesian approach to evaluate the diagnostic performance of a commercially available I-ELISA kit to detect CFT: Complement fixation test; I-ELISA: Indirect enzyme-linked immunosorbent assay; AI: Artificial insemination; ISaBoS: International standard anti-Brucella ovis serum; Se: Sensitivity; Sp: Specificity; γ: Covariance; CrI: Credibility interval.The authors declare that they have no competing interests.JLC and BGB organized the sample collection. BGB conceived and designed the testing study and directed the test performance. YC and AD performed all the immunological analyses and helped design the testing study. AP conceived the model, performed data analyses and drafted the manuscript. AP, JLC, LM, YC, AD and BGB contributed to the conception and design of the whole study, revised the manuscript and approved the final manuscript.Groupement de Défense Sanitaire des Alpes de Haute Provence. LM: MD, PhD, Professor of Public Health at Paris Medical University. YC and AD: BSc. (Biol.). BGB: DVM, PhD, Senior Research Director, Head of the EU/OIE/FAO Brucellosis Reference Laboratory (ANSES).AP: DVM, Teacher and researcher in National Veterinary School of Maisons-Alfort. JLC: DVM, Veterinary practitioner, Veterinary Advisor of the


Cephalopholis argus; Epinephelidae) at 21 locations (N = 550) using DNA sequence data from mtDNA cytochrome b and two nuclear introns .The Coral Triangle (CT), bounded by the Philippines, the Malay Peninsula, and New Guinea, is the epicenter of marine biodiversity. Hypotheses that explain the source of this rich biodiversity include 1) the center of origin, 2) the center of accumulation, and 3) the region of overlap. Here we contribute to the debate with a phylogeographic survey of a widely distributed reef fish, the Peacock Grouper  among five regions: French Polynesia, the central-west Pacific , Indo-Pacific boundary , eastern Indian Ocean (Cocos/Keeling and Christmas Island), and western Indian Ocean . A strong signal of isolation by distance was detected in both mtDNA  and the combined nuclear loci . We detected evidence of population expansion with migration toward the CT. Two clusters of haplotypes were detected in the mtDNA data (d = 0.008), corresponding to the Pacific and Indian Oceans, with a low level of introgression observed outside a mixing zone at the Pacific-Indian boundary.Population structure was significant (Φ5 year glacial cycles, followed by population expansion toward the CT, and overlap of divergent lineages at the Pacific-Indian boundary. This pattern of isolation, divergence, and subsequent overlap likely contributes to species richness at the adjacent CT and is consistent with the region of overlap hypothesis.We conclude that the Indo-Pacific Barrier, operating during low sea level associated with glaciation, defines the primary phylogeographic pattern in this species. These data support a scenario of isolation on the scale of 10 Current efforts to identify and preserve biodiversity are dependent upon our ability to locate hotspots and to understand how that diversity is generated. Conservation efforts must preserve not just standing biodiversity but also the mechanisms that produce it . The CorThe center of origin hypothesis was proposed by Ekman , who sugIn contrast, the center of accumulation hypothesis  proposesContemporary species distributions are the most common line of evidence offered to examine these hypotheses yet no consensus has evolved. Mora et al.  examinedPomacentrus moluccensis) .Sequences for each locus were aligned, edited, and trimmed to a common length using the DNA sequence assembly and analysis software HASE 2.1 ,45 as imHASE 2.1 . We condHASE 2.1  and depoC. argus, including haplotype diversity (h) and nucleotide diversity (π), were estimated with algorithms from Nei [ARLEQUIN 3.5 [t-tests using the online calculator GraphPad . The AIC implemented in jMODELTEST 0.1.1 indicated the TPM1uf+G as the best-fit model of DNA sequence evolution with a gamma value of 0.065. Median-joining networks were constructed using the program NETWORK 4.5 with default settings [RAXML 7.2.7 [b sequences of two congenerics (C. urodeta and C. taeniops) obtained from GenBank . Bootstrap support values were calculated using default settings with 1000 replicates. The ML tree topology was confirmed by neighbor-joining (NJ) and Bayesian Markov Chain Monte Carlo (MCMC) analysis using MEGA 4.0 [MRBAYES 3.1.1 [d) in ARLEQUIN.Summary statistics for from Nei  as impleQUIN 3.5 . To testsettings . An intrML 7.2.7 . Trees wMEGA 4.0  and MRBAES 3.1.1 , respectES 3.1.1  and a gaARLEQUIN. To determine confidence intervals around this value we calculated Harpending's raggedness index, r [FS [FS indicate an excess of low-frequency haplotypes, a signature characteristic of either selection or a recent demographic expansion [To determine whether the number of pairwise differences among all DNA sequences reflected expanding or stable populations , we calcindex, r , which tx, r [FS , which ixpansion .C. argus, an analysis of molecular variance (AMOVA) was performed in ARLEQUIN using 20,000 permutations. Because the TPM1uf+G model of sequence evolution is not implemented in ARLEQUIN, we used the most similar model available [FST (ΦST), which incorporates the model of sequence evolution, was calculated for the entire dataset and for pairwise comparisons among all locations. We maintained α = 0.05 among all pairwise tests by controlling for the false discovery rate as recommended by Benjamini and Yekutieli [ST values and geographic distance using the Isolation-by-Distance Web Service 3.16 [ST values and again with negative ΦST values converted to zeros.To test for hierarchical population genetic structure in vailable  with a gekutieli  and reviekutieli . A Manteice 3.16 . Mantel BEAST 1.5.4 [MODELTEST) [TRACER 1.5 [To estimate the time to coalescence we used the Bayesian MCMC approach implemented in ST 1.5.4 . We condDELTEST) ,62 and rACER 1.5 .HO) and expected heterozygosity (HE) were calculated for each locus and an exact test of Hardy-Weinberg equilibrium (HWE) using 100,000 steps in a Markov chain was performed using ARLEQUIN. To test whether HE differed between ocean basins we calculated unpaired t-tests as described above. Linkage disequilibrium between the two nuclear loci was assessed using the likelihood ratio test with 20,000 permutations in ARLEQUIN. We tested for population expansions by calculating Fu's FS [ARLEQUIN. Genotypes for each individual at the GnRH and S7 introns were compiled and used to calculate FST for the multi-locus dataset and for pairwise comparisons between locations in ARLEQUIN. The false discovery rate among multiple comparisons was controlled as described above. Median-joining networks for alleles at each locus were constructed using the program NETWORK. We tested for correlation between pairwise FST values and geographic distance (isolation-by-distance) among all populations using the Isolation-by-Distance Web Service [Observed heterozygosity  were calculated with the software MIGRATE 3.1.6 [ST that did not significantly differ from zero) into demes defined by region (see Results). This program uses a Bayesian MCMC search strategy of a single, replicated, two million step chain. The default settings for priors were used with an unrestricted migration model. Estimates of the number of immigrants per generation (Nm) were calculated by multiplying final estimates of θ and M [Migration rates between groups  we grouped the specimens from the central Pacific locations of Samoa and Tokelau, and Baker and Howland Island. The number of individuals (N), number of haplotypes (Nh), haplotype diversity (h), and nucleotide diversity (π) for each location are provided in Table C. argus was low (π = 0.005) while the corresponding haplotype diversity was high (h = 0.80). Across all samples, π = 0.001 - 0.009 and h = 0.38 - 0.96 with higher genetic diversity detected in the Pacific compared to the Indian Ocean . Using the program BEAST and implementing a molecular clock of 2% per million years [d = 0.008, Figure We resolved a 729 bp segment of cytochrome ls Table . Due to on years -69 we esST was 0.297 (P < 0.001). When we grouped samples by ocean basin (as described in Methods) we found significant structure between the Pacific and Indian Oceans . Within oceans we found low but significant structure in the Pacific Ocean  and higher structure in the Indian Ocean . Pairwise comparisons indicate low levels of structure at the eastern edge of the range distinguishing Marquesas and Moorea , a result that indicates a stable population. In contrast, Fu's FS resulted in FS = -12.8 (P < 0.001) signifying an excess of low-frequency haplotypes and an expanding population. Grouping locations that demonstrated no significant population structure , CW  and WI  but no evidence for population expansion was found for either IB  or EI .Population expansion parameters for the overall dataset gave conflicting results. As expected with the presence of two divergent mitochondrial lineages, the mismatch distribution for the overall dataset was bimodal Figure  and resuN), number of alleles (Na), observed heterozygosity (HO), expected heterozygosity (HE), and the corresponding P-value for the exact tests for HWE are listed in Table P = 0.002 and 0.044, respectively) while the sample from Diego Garcia was found to have an excess of heterozygotes at the S7 locus (P = 0.047) . There was no indication of linkage disequilibrium between the two loci (P > 0.05).We resolved 245 bp of the GnRH intron in 488 specimens and 393 bp of the S7 intron in 490 specimens Table . Seven p7) Table . Across FST values for GnRH, S7, and the multi-locus dataset were FST = 0.078 (P < 0.001), FST = 0.099 (P < 0.001), and FST = 0.127 (P < 0.001), respectively. Analyses of these data reveal patterns of population structure that are concordant with the mitochondrial dataset. Grouping samples by ocean basin (as above) revealed significant structure between the Pacific and Indian Oceans  and significant structure within ocean basins . Pairwise FST for the multi-locus dataset are reported in Table Overall P = 0.082; S7, r = 0.12, P = 0.235; data for geographic groups not shown). Fu's FS calculations offered no evidence for expanding populations for either the overall dataset  or for the geographic groups (data not shown).As might be expected from loci with low numbers of closely related alleles, the mismatch distributions for the overall nuclear dataset and for the five geographic groups  were unimodal and resulted in non-significant raggedness indices  contribute to genetic diversity across the central portion of the range , they rarely receive migrants  and geographic distance in the mtDNA  and the multi-locus nuclear  datasets. Replacing negative values of ΦST and FST with zeros did not affect the pattern or statistical significance. To test if genetic structure between ocean basins was driving IBD we conducted Mantel tests within oceans and found weaker but still significant correlations between genetic distance and geographic distance with the Cytb dataset  but not the multi-locus nuclear dataset .Mantel tests showed a strong correlation between genetic distance  has fostered numerous and seemingly conflicting hypotheses. The center of origin hypothesis postulates that elevated rates of speciation in the CT have resulted in high species diversity . In contCephalopholis argus demonstrates significant levels of genetic structure that indicate a historical partition between the Pacific and Indian Oceans (Table d = 0.008) indicating isolation for approximately one million years , a time interval that corresponds to Pleistocene sea level fluctuations linked to Milankovitch climate cycles on the scale of 105 years [ns Table . Two mit05 years . Our anaWhile incomplete lineage sorting is a serious problem for species level reconstructions, our pattern of divergence across the IPB is corroborated by three independent markers. Additionally, the finding of isolation by distance across the species range is strong evidence that the patterns we present here are not driven by stochastic events.The Sunda shelf, surrounding the Malay Peninsula and western islands of Indonesia, and the Sahul shelf off northern Australia and New Guinea, separate the Pacific and Indian Oceans and together are known as the Indo-Pacific Barrier (IPB) . Over thC. argus. This species demonstrates strong population structure between Pacific and Indian Ocean locations in both the mitochondrial and nuclear datasets. The mismatch distribution for C. argus is distinctly bimodal  and French Polynesia (FP) toward the center of the range . Other irostris ). Pacifi berndti ; Naso vllamingii ; Nerita lbieilla ). C. arge Figure  while poe Figure , 5.There is compelling evidence for the influence of the IPB on coral reef organisms from intraspecific lineage sorting to species level distributions. The degree of range expansion or lineage mixing after the last glacial maximum varies among taxa and may reflect species level differences in dispersal ability, reproductive strategy, competitive ability, or habitat requirements.C. argus demonstrates no population structure across the nearly 8,000 km central Pacific range, from Kiritimati to Palau. However, pairwise ΦST and FST values and migration rates indicate that populations at the eastern end of the range at Moorea and the Marquesas are isolated. This pattern of extensive population connectivity across the central Pacific with isolation at the ends of the Pacific range is emerging in reef fishes .C. argus along an east-west gradient and indicate unidirectional dispersal out of the WIO. Despite being geographically located in the Indian Ocean, the eastern Indian Ocean faunas at Cocos/Keeling and Christmas Islands, and Western Australia are closely affiliated with the Pacific ichthyofauna with only 5% of reef fishes at Cocos/Keeling of exclusively Indian Ocean origin [Lutjanus kasmira [C. argus, this study) indicate that Diego Garcia is a part of the Western Indian Ocean Province as described by Briggs [The isolation of the western Indian Ocean (WIO) is supported by both species distributions  and intrn origin . Insteadn origin ,22. Diegn origin . The dis kasmira  and C. ay Briggs . While wCephalopholis argus indicates that this species was strongly impacted by Pleistocene sea level fluctuations which resulted in the partitioning of this species into Pacific and Indian Ocean mitochondrial lineages that are distinguished by fixed differences (d = 0.008). Following the end of the last glacial maximum, connectivity between the Pacific and Indian Oceans resumed and C. argus populations expanded. Representatives of each mitochondrial lineage are now found in both oceans with the center of diversity occurring in the Coral Triangle: a pattern that we offer as support for the region of overlap hypothesis. In a recent review 15 out of 18 species demonstrated significant structure across the IPB, such that subsequent contact would constitute support of the region of overlap hypothesis [Our genetic survey of the grouper pothesis . HoweverMRG obtained samples from the central Pacific and the Philippines, participated in DNA sequencing, data analysis, data interpretation, and prepared the manuscript. BWB participated in study design, funding, and sampling from the Pacific and Indian Oceans, and helped prepare the manuscript. TRB participated in DNA sequencing. LAR obtained samples from across the Pacific and Indian Oceans, and helped prepare the manuscript. SJN obtained samples from Western Australia and the eastern Indian Ocean. LAG and LVH participated in sequencing individuals from Western Australia and the east Indian Ocean. MTC obtained samples from throughout the Pacific and Indian Oceans, participated in the design and coordination of the study, and helped prepare the manuscript. All authors approved the final manuscript.


Brassica napus L. can be used to broaden the genetic diversity and to develop a heterotic genepool for rapeseed hybrid breeding. Domesticated vegetable types are usually employed as B. oleracea parents. We sought to evaluate the potential of wild species as parents for Resyn lines. Fifteen Resyn lines were derived by crossing wild B. oleracea ssp. oleracea and oilseed B. rapa, and 29 Resyn lines were generated from 10 wild Brassica species . Genetic distances were analyzed with AFLP markers for 71 Resyn lines from wild and domesticated B. oleracea, and compared with 55 winter, spring, vegetable, and Asian B. napus genotypes. The genetic distances clearly showed that Resyn lines with wild species provide a genetic diversity absent from the breeding material or Resyn lines from domesticated species. Forty-two Resyn lines were crossed with one or two winter oilseed rape testers, resulting in 64 hybrids that were grown in one year and four locations in Germany and France. The correlation between hybrid yield and genetic distance was slightly negative (r = −0.29). Most of the hybrids with Resyn lines from wild B. oleracea were lower in yield than hybrids with Resyn lines from domesticated B. oleracea. It is promising that Resyn lines descending from unselected wild B. oleracea accessions produced high-yielding hybrids when crossed with adapted genotypes: these Resyn lines would be suited to develop heterotic pools in hybrid breeding.Resynthesized (Resyn) The online version of this article (doi:10.1007/s00122-012-2036-y) contains supplementary material, which is available to authorized users. Brassica napus L. is an allopolyploid (amphidiploid) species that resulted from interspecific hybridization between B. oleracea (C genome) and B. rapa (A genome). Allopolyploidy is an evolutionary process whereby two or more genomes are combined by spontaneous inter-specific or inter-generic hybridization, followed by chromosome doubling. Several important crops, such as bread and durum wheat, oat, cotton, and coffee, are allopolyploid (Feldman and Levy B. napus it is possible to produce ‘resynthesized’ (Resyn) genotypes via an artificial cross between the parental species B. oleracea and B. rapa. Resyn rapeseed genotypes have been used for many years to broaden the genetic variation of oilseed rape: an overview of this strategy of introgression of single traits is given by Qiong et al. . All of them have the same chromosome number (n = 9), can be crossed with each other, and produce fertile hybrids  analyze the genetic distances in collections of winter, spring, vegetable, and Asian B. napus genotypes (n = 55) in comparison to 71 Resyn lines, of which 44 originated from crosses with wild B. oleracea taxa and (2) evaluate the yield of hybrids derived from crosses of 42 Resyn lines with one or two male sterile B. napus tester lines.In the present study, Resyn lines were obtained from crosses of B. napus varieties , and obsolete varieties including ‘Mansholts Hamburger’ and ‘Zachodni’. Chinese, Turkish, and vegetable B. napus varieties completed the broad genetic material.The collection of 126 genotypes consisted of 55 DOMn = 27) and wild Resyn lines (RWILDn = 44), according to the origin of their B. oleracea parental genotypes (ESM 2). The RDOM lines originated from crosses of diverse domesticated B. oleracea taxa (vegetable forms) that were described earlier by Girke et al.  with 11 wild B. oleracea taxa (C genome). Depending on the C genome donator, the group of RWILD lines was further subdivided into RWTYPE and RWSPEC Resyn lines. Twenty-nine RWSPEC lines originated from crosses of B. rapa with ten wild species of the B. oleracea or C genome group. The names of the RWSPEC lines were composed of their parental genotypes, where the first two letters describe the paternal species: B. bourgaei (BO), B. cretica (CR), B. incana (IN), B. insularis (IS), B. hilarionis (HI), B. macrocarpa (MA), B. montana (MO), B. rupestris (RU), B. taurica (TA), and B. villosa (VI). The last letter stands for the B. rapa genotype: ‘Y’ for ‘Yellow Sarson’ (spring oilseed) or ‘L’ for ‘Largo’ (winter oilseed). Fifteen RWTYPE lines were obtained from crosses with B. oleracea ssp. oleracea (wild B. oleracea) with winter B. rapa oilseed variety ‘Largo’ and breeding line ‘NPZ 00’. The names of the RWTYPE lines are composed of the letter ‘J’ and a number (ESM 2). Taxonomic classification was carried out according to Hanelt . AFLP procedures  were received from Norddeutsche Pflanzenzucht Hans-Georg Lembke KG and Syngenta Seeds, respectively. In the 2008–2009 season, hybrids with ‘Tester B’ were produced in the field in Biemsen, Germany, with ‘Tester A’ in Malchow, Germany, and with both testers at the Georg-August-University Göttingen in the greenhouse. In the field, hybrid seeds were produced as described by Girke et al. , with miSixty-four test hybrids and three check varieties were grown in a randomized block design without replication at four locations during the 2009–2010 growing season. Plot sizes differed from 11.25 to 18.0 m². The trials were performed at Hohenlieth , Einbeck and Göttingen , and Resson sur Metz . Due to seed shortage, a reduced set of 39 hybrids was tested at five additional locations . The results of these trials appear in ESM 3, while the data from the larger hybrid test set are presented throughout this publication. The following check varieties were included: hybrid variety ‘Visby’, ‘fertile line tester A’, and ‘fertile line tester B’.−1 (relative to 91 % dry matter). As quality parameters of the harvested seeds, the oil , protein , and glucosinolate  content were measured using near-infrared reflectance spectroscopy , development before and after winter , plant height at the end of flowering (in cm), and lodging . Winter hardiness was estimated as the difference between the scores of before and after winter development, with smaller numbers indicating better winter hardiness. Seed yield was determined in dt haH²), and tests of significance were performed using PLABSTAT software (Utz Yij = μ + gi + lj + (gl)ij, where Yij is defined as the observation of genotype i at location j, μ is the overall mean, gi is the effect of genotype i , lj is the effect of location j , and (gl)ij is the corresponding interaction effect, including experimental error. The ANOVA was calculated as a mixed model, where the effect of the location was considered random and the effect of the genotype was considered fixed.The statistical analysis of the field trials, estimations of correlations, least significant differences, heritability , and RDOM and RWILD lines, were higher than the within-group distances. The highest mean between-group genetic distances involved Resyn lines (0.61–0.62), whereas the smallest genetic distance was observed between winter and vegetable B. napus (0.34). All genetic group comparisons with RDOM lines revealed higher mean genetic distances (0.45–0.53) than those with B. napus varieties . The mean within-group distance in B. napus varieties was lower than that in Resyn lines: 0.29 in winter, 0.28 in spring, 0.37 in Asian, and 0.41 in vegetable B. napus.The mean genetic distance of all 15,750 paired comparisons was 0.54, with a range of minimum 0.09 and maximum 0.81 in all pairwise comparisons Table . The genB. napus occurred between ‘Bronowski’ and ‘Zachodni’ (0.44), whereas ‘Golden’ and ‘Licosmos’ were associated with a genetic distance of only 0.19. In the group of Asian B. napus, the ‘Italy’ and ‘Xiangyou 11’ varieties revealed the smallest (0.29) genetic distance, while ‘Linyou 5’ and ‘Ganyu 3’ had the highest genetic distance (0.43). The variation in the B. napus vegetable forms ranged from 0.23 (between ‘Brauner Schnittkohl’ and ‘Goldgelber Zarter Butter’) to 0.50 (between ‘Grüner Schnittkohl’ and ‘Wilhelmsburger Steckrübe’). The highest within-group variation occurred in the RWILD lines. The RWILD lines ‘MAY 1’ and ‘MOY 1’ had the highest genetic distance (0.79), while lines ‘J166’ and ‘J400’ had the smallest genetic distance (0.09) within this group. All pairwise comparisons are available by request.The winter oilseed rape varieties ‘Billy’ and ‘Oase’ had the smallest genetic distance (0.12), while varieties ‘Campari’ and ‘Mansholt 54’ had the highest genetic distance (0.42). The highest variation in spring WILD lines. Overall, the group of Resyn lines showed the largest genetic distances, and various subgroups occurred within the RWILD genotypes that can be traced back to the corresponding parental genotypes: RWTYPE, RWSPEC, ‘Yellow Sarson’, and ‘Largo’. Two subgroups were differentiated within the RWSPEC lines , and the other group consisted of crosses of Brassica wild species with the spring B. rapa ‘Yellow Sarson’ (upper right corner). Both groups were distinguished on the basis of the first principal coordinate. Corresponding subgroups were detected within the RWTYPE Resyn lines , and all vegetables, with the exception of the ‘Wilhelmsburger Steckrübe’, which was located in cluster IVa. Three Turkish varieties formed cluster II with the spring B. napus . The Asian genotypes formed a separate cluster (cluster III).The pattern of genetic distances within and between genotype groups is shown in more detail in the dendrogram from the cluster analysis Fig. . Here, tus Figs. , a patteWSPEC lines, with the C genome of wild Brassica species. Clusters VIa, VIb, and VIc separated at mean genetic distances of 0.60, 0.63, and 0.65, respectively. Cluster VIa included RWSPEC genotypes and two RDOM lines (‘FS 94.3’ and ‘R 76’), whereas cluster VIb integrated three RDOM lines  and two RWTYPE lines (‘OLY 1’ and ‘OLY 2’), which were obtained from crosses with B. oleracea ssp. oleracea. Most RWTYPE lines were located in clusters Va and Vb. Cluster Va was separated from the B. napus varieties at a genetic distance of 0.55, while cluster Vb formed a subgroup with the RDOM lines in cluster IVa, separated at a genetic distance of 0.58. The majority of the RDOM lines grouped in clusters IVa and IVb, which were located between the subgroups of the varieties  and the RWSPEC lines (cluster VI), which were obtained from crosses with wild Brassica species. RDOM line ‘H 149’ was positioned adjacent to cluster IVa, while the rest of the RDOM lines were assigned to clusters I (winter B. napus) and VI (RWSPEC).Clusters I and II, containing the winter and spring genotypes, respectively, were separated at a mean genetic distance of 0.37, while clusters II and III (Asian group) had a genetic distance of 0.41, respectively. Clusters VIa, VIb, and VIc showed the highest distances to the cultivated genotypes in clusters I, II, and III. These clusters included all RP = 0.01) for the test hybrids for all traits except winter hardiness .Genotype and location were significant sources of variation (ss Table . The her−1) and ‘fertile line tester B’ (33.7 dt ha−1) were lower than the mean yield of the test hybrids (39.2 dt ha−1), while the yield of check variety ‘Visby’ exceeded all test hybrids  or RWSPEC (36.5 dt ha−1) lines . The yield of hybrid ‘Tester A × H 176’ was nearly as high as that of the check variety ‘Visby’: ‘H 176’ is an RDOM genotype  was estimated for yield from the resulting hybrids.The mean seed yield of hybrids originating from RWTYPE and RWSPEC lines ranged between 40 and 43 % (Table DOM lines. ‘Fertile line tester B’ had a low oil content (42.1 %) compared with ‘fertile line tester A’ (45.4 %) and ‘Visby’ (43.6 %). Three test crosses of ‘Tester A’ and RDOM lines ‘H 149’, ‘H 176’, and ‘S 13’, with oil contents of 45.5, 45.6, and 46.2 %, respectively, exceeded the ‘high-oil’ line ‘fertile line tester A’. The mean protein content of the test crosses with Resyn lines was higher than the mean of check varieties, with the highest protein content in ‘Tester A × MOL 2’ (21.0 %). Erucic acid and glucosinolate contents varied significantly in the test hybrids  and in the winter, spring, Asian, and vegetable subgroups  corroborated the results of earlier studies. Becker et al.  estimatee et al.  reportedDOM and RWILD subgroups, was higher than in the genotype groups of varieties, indicating a reduction in genetic variation as a result of breeding and selection. This decrease in genetic variation between Resyn lines and varieties was previously reported by studies including Resyn lines . Becker et al. , which may explain why the genetic distance to the Asian gene pool was lower than the distance to the European gene pool  than the variation among the variety genotype groups.In accordance with Girke et al. , the RDOWILD lines harbor a genetic diversity not present in the breeding material or the RDOM lines. RWILD lines would therefore be suitable to form a heterotic gene pool for hybrid breeding that is adequately distant from the adapted B. napus winter oilseed rape.Our genetic distance data indicate that the Rr = −0.29 (P = 0.1). This result agrees with earlier studies: Girke et al. (r = −0.01) for hybrid yield and genetic distance in 12 hybrids of the spring variety ‘Korall’ and Resyn lines, while Girke et al.  and hybrid yield, as well as genetic distance and heterosis, are correlated positively. Charcosset and Essioux  and Melcr = 0.59 between hybrid yield and genetic distance in diallelic crosses of seven spring oilseed varieties, and Riaz et al.  in crosses between four elite cotton lines and 12 exotic germplasm lines. In crosses within and between groups of American and Chinese maize lines, related lines showed the highest correlations between genetic distance and grain yield (r = 0.47), while inter-group hybrids were not correlated Below is the link to the electronic supplementary material.


T5 is a novel splice variant of heparanase, an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains at a limited number of sites. T5 splice variant is endowed with pro-tumorigenic properties, enhancing cell proliferation, anchorage independent growth and tumor xenograft development despite lack of heparan sulfate-degrading activity typical of heparanase. T5 is over expressed in the majority of human renal cell carcinoma biopsies examined, suggesting that this splice variant is clinically relevant. T5 is thought to assume a distinct three-dimensional conformation compared with the wild type heparanase protein. We sought to exploit this presumed feature by generating monoclonal antibodies that will recognize the unique structure of T5 without, or with minimal recognition of heparanase, thus enabling more accurate assessment of the clinical relevance of T5. We provide evidence that such a monoclonal antibody, 9c9, preferentially recognizes T5 compared with heparanase by ELISA, immunoblotting and immunohistochemistry. In order to uncover the clinical significance of T5, a cohort of renal cell carcinoma specimens was subjected to immunostaining applying the 9c9 antibody. Notably, T5 staining intensity was significantly associated with tumor size (p = 0.004) and tumor grade (p = 0.02). Our results suggest that T5 is a functional, pro-tumorigenic entity. Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPGs) presumably at sites of low sulfation, releasing saccharide products with appreciable size (4–7 kDa) that can still associate with protein ligands and facilitate their biological potency wt) heparanase protein. We sought to exploit this presumed feature by generating monoclonal antibodies that will recognize the unique structure of T5 without, or with minimal recognition of heparanase, thus enabling more accurate assessment of the clinical relevance of T5. Here, we provide evidence that such a monoclonal antibody (mAb), 9c9, preferentially recognizes T5 compared with heparanase by ELISA, immunoblotting and immunohistochemistry. A cohort of renal cell carcinoma specimens was subjected to immunostaining applying mAb 9c9. Notably, T5 staining intensity was significantly associated with tumor size (p = 0.004) and tumor grade (p = 0.02), suggesting that T5 is a functional, pro-tumorigenic entity.Alternative splicing increases the coding capacity of the genome, generating multiple proteins from a single gene. The resulting protein isoforms frequently exhibit different biological properties that may play an essential role in tumorigenesis GGAATTCATGCTG CTGCGCTCG 3′ and reverse 5′AACTGCAGTCATTTCTTACTTGAGTAGG 3' and was inserted into bacterial expression vector . The expression and purification of MBP and MBP-T5 was carried out according to the manufacture's (NEB) instructions. Briefly, MC1061 bacteria culture was grown in the presence of isopropyl-β-D-thiogalactopyranoside  for 5 h at 16°C. Bacteria were then harvested by centrifugation ; the pellet was re-suspended in column buffer  and incubated with lysosyme  for 30 min on ice. Following sonication and centrifugation, , the supernatant was mixed gently with 1/10 volume of amylose resin . The resin was then packed in a column, washed, and MBP-T5 was eluted with column buffer containing 10 mM maltose. Elution fractions were analyzed by SDS-PAGE and protein concentration was determined by the Bradford assay (Bio-Rad).T5 was amplified by PCR using the following primers: forward 5' BALB/c mice were immunized with 50 µg of MBP-T5 fusion protein in complete Freund's adjuvant , followed by five injections of MBP-T5 (50 µg) in incomplete Freund's adjuvant (IFA) every 2 weeks. Splenocytes were then isolated, fused with NSO myeloma cells and hybridomas were screened for their ability to bind MBP-T5 or heparanase but not MBP by ELISA, essentially as described Preparation of cell lysates and immunoblotting analyses were carried out as described previously The study included 66 patients with renal cancer  that werStaining of formalin-fixed, paraffin-embedded 5 micron sections was performed essentially as described Immunofluorescent double staining of heparanase and T5 applying antibody #733 and mAb 9c9 was carried out essentially as described Univariate association between T5 staining intensity and clinical and pathological findings as well as patients outcome, were analyzed using Chi Square tests .We have recently reported the cloning of T5, a functional splice variant of heparanase endowed with pro-tumorigenic properties In order to reveal whether our antibodies are suitable for immunohistochemical analysis, and to explore their specificity, tumor xenografts were subjected to immunostaining. Remarkably, the specificity observed by ELISA and immunoblotting was also seen in immunostaining. Thus, antibody 7c4 reacted similarly with tumor xenografts generated by CAG human myeloma cells over-expressing heparanase or T5 , left. IWe have next examined the capability of mAb 9c9 to detect T5 in tumor biopsies. In head and neck cancer, mAb 9c9 stained positively the epithelial compartment of some carcinomas ; in otheWe employed mAb 9c9 to examine T5 expression in specimens collected from RCC patients. Sixty six patients  were included in the study (median age at diagnosis was 60.3±13.3), predominantly classified as clear cell carcinoma (75%). Demographic and clinical description of patients is shown in Alternative splicing considerably expands the information content of the transcriptome and proteome through the expression of different mRNAs from a single gene. Splicing abnormalities are a common characteristic of cancer, including genes associated with cell growth, motility, apoptosis, and response to chemotherapy We have recently described a human heparanase splice variant termed T5 Taken together, we provide evidence that T5 is a clinically relevant splice variant which may complement the pro-tumorigenic function of heparanase. T5 likely assumes a distinct three-dimensional folding other than that of heparanase, a notion that is now supported by our mAb 9c9. Expression of T5 in blood vessels and immune cells ; 3D may Figure S1ELISA. 96-well plate was coated with MBP (▪), MBP-T5 (▪), or heparanase (▪) proteins (1 µg/ml) and incubated with the indicated mAb for 2 h at room temperature. Following washes, anti-mouse IgG HRP-conjugated secondary antibody  was applied and mAb binding was visualized by colorimetric reaction (TMB/H2SO4). Shown are representative OD values obtained.(TIF)Click here for additional data file.Figure S2A. Schematic diagram of heparanase/T5 structure. Heparanase is first synthesized as a pre-proenzyme, harboring 35 amino acids signal peptide  which is removed upon entering the ER. The protein is then subjected to glycosylation and secreted as a ∼65 kDa latent protein (upper panel). Proteolytic processing removes the linker domain (Ser110–Gln157), resulting in 8 kDa (Gln36–Glu109), and 50 kDa (Lys158–Ile543) protein subunits (second panel) that heterodimerize to yield an active enzyme. Replacement of the linker segment with three pairs of glycine (G)-serine (S) results in a constitutively-active single chain enzyme . The SP, 8 kDa and linker fragments are retained in T5, but the 50 kDa subunit is excised except for 9 amino acids, which are followed by the addition of three unique amino acids . B. Epitope determination. HEK 293 cells were transfected with wild type 8 kDa gene construct or 8 kDa deleted at its C-terminus  or N-terminus . Control cells were transfected with an empty plasmid (Vo). Lysate samples were then subjected to immunoblotting applying mAb 5B5 (upper panels) or mAb 9D5 (second panels). Equal protein loading is exemplified by actin immunoblotting (fourth panel); Myc-tag immunoblotting confirms comparable expression levels of gene constructs (third panel). The epitope of both antibodies is localized at the protein N-terminus.(TIF)Click here for additional data file.Table S1Performance of anti-T5 monoclonal antibodies.(TIF)Click here for additional data file.


Vitis vinifera L., East Asian and North American Vitis species and hybrids, as well as 2 varieties of muscadine grapes were estimated by HPLC-MS/MS. There were 45 anthocyanins, 28 flavonols, 8 flavan-3-ols, 9 cinnamic acids, 5 benzoic acids, 5 ellagic acids and 2 stilbenes detected in all the samples. Total contents of each phenolic type varied significantly among the different grape cultivars investigated. There was also a large variability in the phenolic compositions of different grape groups. The differences in anthocyanin composition were obvious between V. vinifera and non-V. vinifera grapes and also between the grapes originating from Eurasia and North America. Quercetin-3-glucuronide and quercetin-3-glucoside were marker flavonol compounds for Euvitis grape skins. Flavan-3-ol monomers were dominant in the skins of muscadine and non-V. amurensis East Asian grapes, whereas polymers were more common in V. vinifera and North American grapes. The muscadine grapes were very rich in flavonols, flavan-3-ols and ellagic acids. Via principal component analysis, these grape cultivars were clustered into three groups according to their characteristic phenolic content and composition.In order to analyze and compare the phenolic characteristics of red wine grapes with diverse genetic backgrounds, skin phenolics among 21 different cultivars belonging to  Vitis L.), which has a long history of cultivation and utilization, is one of the most important commercial fruit crops worldwide. There are more than 70 grape species and a large number of grape cultivars growing all over the world [Vitis L. is divided into two subgenera: Euvitis Planch. and Muscadinia Planch. Based on geographical distributions, there are three groups of Euvitis species, including V. vinifera, East Asian and North American Vitis species. Muscadinia Planch., which refers mainly to V. rotundifolia Michx., is originated in the southeastern United States. This grape is also called muscadines and is genetically distinct from Euvitis species [The grape , two phenolic rings (stilbenes), three rings  and complex ring (ellagic acids) . Anthocybilities –6.V. vinifera [Liquid chromatography-tandem mass spectrometry (HPLC-MS) technology has become a popular tool for the efficient identification of phenolic compounds. As a result, a lasrge amount of phenolic compounds have been identified and quantified in the grape berries and wines from vinifera –9, muscavinifera –12 and svinifera ,14. HoweV.labrusca “Catawba” and “Niagara Rosada” (1457.04 and 1065.63 μg MGE/g DM), which were native to North America. Among the East Asian grapes, V. amurensis “Zuoshanyi”, “Zuoshaner” and V. ficifolia “Sangye”, and a V. amurensis × V. vinifera hybrid “Zuohongyi”, produced high amounts of anthocyanins in their skins (more than 10000 μg MGE/g DM). Meanwhile, V. dividii “Black Pearl”, V.xunyangensis “Mi” and V.quinquangularis “Mao” had low amounts of TAs (1000–2000 μg MGE/g DM).Total anthocyanins (TAs) of all the samples ranged from 1065.63 to 16840.99 μg Malvidin-3-glucoside (MGE) /g dry weight (DM) . There wV. vinifera (77.8%) and East Asian species (53.55%), in which mv-3-glucoside (glc) and mv-3,5-diglc were, respectively, the most anthocyanin. The chief anthocynin types in most North American grapes were cy- (43.21%) and dp- derivatives (32.20%) due to their high levels of cy-3-glc and dp-3-glc. In the case of V. rotundifolia grapes, only the diglucosides of the six anthocyanidins were found, where dp-3,5-glc was the most abundant anthocyanin in “Alachua” (45.16%) and cy-3,5-glc was the most abundant in “Noble” (36.17%). Moreover, pt-3,5-glc (21.44% on average) was relatively abundant in the skins of both cultivars. In the skins of the hybrids, anthocyanins were primarily represented by mv-, dp- and pt-derivatives, but the proportions were different in Euro-Asian hybrids  and Euro-American hybrids . Small amounts of pg-3,5-diglucosides were detected only in V. rotundifolia “Alachua” and “Noble”, and pg-3-glucoside was detected in V.labrusca “Niagara Rosada” and V.aestivalis “Black Spanish”.A total of 45 anthocyanin compounds were identified from all the samples, including monoglucoside, diglucoside and their acylated derivatives of six anthocyanidins: delphinidin (dp), cyanidin (cy), petunidin (pt), pelargonidin (pg), peonidin (pn) and malvidin (mv) . The mv-V. vinifera grapes, East Asian grapes, and Euro-Asian and Euro-American hybrids, accounting for 87.57% of TA on everage. Muscadine and North American grapes were different, where 3′,4′- and 3′,4′,5′-substituted anthocyanins accounted for 52.93% and 45.89% of TAs, respectively, and 4′-substituted anthocyanins were minor components.According to the numbers of B-ring substituents of anthocyanidins , the antV. vinifera grapes (94.22%) > East Asian grapes (77.30%) > Euro-American hybrids (72.49%) > Euro-Asian hybrids (49.10%) > muscadine grapes (42.86%) > North American grapes (24.23%).Anthocyanins can also be classified into non-methylated  and methylated ones . The proportions of methylated anthocyanins in the TAs of the skins decreased in the order: V. vinifera grapes only contained the monoglucoside anthocyanins, but V. rotundifolia grapes only had diglucosides anthocyanins. Among the East Asian species and V. amurensis × V. vinifera hybrids, diglucosides anthocyanins were dominant in most grapes (accounting for 86.81% of TAs on average) except V.quinquangularis “Mao” and the hybrids “Zuohongyi” and “Hasang”. While monoglucoside anthocyanins were the main anthocyanins in North American grapes and Euro-American hybrids (accounting for 74.76% of TAs on average), except V.aestivalis “Black Spanish” and the hybrid “St. Croix”.With regard to monoglucoside and diglucosides anthocyanins, there was a distinct separation among the different species. The p-coumaroyl derivatives were the most abundant ones with a high propotion (21.61% of TAs on average) in most samples with the exception of V. amurensis and V. rotundifolia grapes, in which no acylated anthocyanins were detected. The percentage of acetyl derivatives was high in the two V. vinifera grapes, but very low in non-V. amurensis East Asian grapes, North American grapes, and Euro-Asian and Euro-American hybrids.Among acylated anthocyanins, the et al. (2008) concluded malvidin derivatives were the most abundant anthocyanins in the majority of germplasms, and monoglucoside derivatives were in V. vinifera and both mono- and di-glucoside derivatives in other Vitis germplasms. Their data were based on the analysis of the anthocyanin composition in the skins of 110 Euvitis grape cultivars [Colored anthocyanins play an important role in the quality of red grapes and wines, which make the anthocyanin profile a major index for classifying grape cultivars and wine. In anthocyanin biosynthesis, there are three branches from naringenin: 4′-substituted, 3′,4′-substituted and 3′,4′,5′-substituted anthocyanins. The latter two have an absolute advantage among grape anthocyanins, but their proportion of TAs largely depends on the expression level of flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase. Meanwhile the expression levels of methyltransferase, glucosyltransferase and acetyltransferase directly influence the degree of methylation, glucosylation and acylation . As a reultivars . For musultivars .V. vinifera and East Asian grapes native to Eurasia, the proportion of which was significantly higher than in North American and muscadine grapes native to American. ii) Mono-glucosides were the only anthocyanin type in European grape skins and dominant in North American grape skins, while di-glucoside anthocyanins were the only type in muscadines and dominant in East Asian grapes. iii) The proportion of acetyl anthocyanins was significantly higher in V. vinifera grapes than in non-V. vinifera grapes.In the present study, we found some interesting trends worth investigating in further research in the molecular mechanism differences of anthocyanin biosynthesis in red wine grapes with various genetic backgrounds and originations. i) Methylated and 3′,4′,5′-substituted anthocyanins were dominant in the skins of V. rotundifolia “Noble” (1892.53 μg QE/g DW) and “Alacha” (1856.99 μg QE/g DW) had almost 2-fold higher TFO contents than the highest bunch grapes which were V. ficifolia “Sangye” (1358.17 μg QE/g DW), V. labrusca “Concord” (200.41 μg QE/g DW), and “Niagara Rosada” (1024.86 μg QE/g DW). Most V. amurensis cultivars  and the French hybrid “Marechal Foch” had less than 200 μg QE/g DW.Flavonols convert from dihydroflavonols through the formation of a double bond between C-2 and C-3 in the dihydroflavonol molecules under the function of flavonol synthase . So the O-glucuronide and quercetin-3-O-glucoside, accounting for 22.49% and 19.01% of TFOs, respectively, were the most common quercetin derivatives in the skins of all cultivars except muscadines, in which quercetin-3-O-rhamnoside (34.81%) and quercetin (26.97%) were the main flavonols. Most V. amurensis cultivars and hybrids were rich in quercetin, ranging from 20%–50% of TFOs, while non-V. amurensis East Asian species were dominated by quercetin-3-O-rhamnoside (42.44% of TFOs on average).Among the 28 detected compounds , there wV. vinifera grapes, in which isorhamnetin, dihydroquercetin, syringetin and kaempferol derivatives accounted for 18.49%, 14.69%, 14.36% and 12.54% of TFOs, respectively.Kaempferol derivatives were the second most important flavonols in the skins of North American grapes and Euro-American hybrids, accounting for 24.04% and 10.19% of TFOs, respectively. However, myricetin derivatives were the second most abundant ones in East Asian grapes, Euro-Asian hybrids and muscadine grapes, accounting for 10.39%, 15.54% and 13.34% of TFOs, respectively. The proportions of other flavonol aglycone derivatives were low in most samples except the two V. vinifera grape skins [Euvitis red grapes. These compounds were not detected in muscadine samples. The large amount of diverse flavonols and dihydroflavonols was an outstanding characteristic of flavonol profiles for the skins of muscadine grapes, which might enhance their antioxidant capacity and make their taste different.Flavonols are related to bitterness, and act as copigments of anthocyanins in the wines . Many prpe skins –24. Our V. labrusca “Niagara Rosada” had the highest TFA content in all the tested samples (1243.67 μg CE/g DW), while V. labrusca “Catawba”, V.aestivalis “Black Spanish”, and the French hybrid “Marechal Foch” had less than 20 μg catechin equivalence (CE) /g DW. The two muscadines also possessed high TFAs (451.48μg CE/g DW for “Noble” and 350.62 μg CE/g DW for “Alachua”). A relatively low TFA contents was found in most East Asian cultivars, and none were detected in most V. amurensis cultivars and its hybrids except “Hasang” and “Changbaijiu” (562.73 and 381.80 μg CE/g DW).Flavan-3-ols mainly contribute to the structure of wines. Flavanol polymers (condensed tannin) play a particularly important role in the astringency of wines. They may react with anthocyanins through an intermolecular copigmentation process, leading to the definition and stability of color in red wines . There wV. vinifera grapes, North American grapes and Euro-American hybrids, the flavan-3-ols were mainly comprised of polymers, accounting for 70%–100% of TFAs, except in the French hybrid “Marechal Foch”. However, monomers were the chief flavan-3-ol type in the muscadines and non-V. amurensis East Asian species. The most abundant monomers were found in V. rotundifolia “Noble” and “Alachua” . Among all the flavan-3-ols, the procyanidin dimer was the most important compound in the non-muscadine grapes. The high levels of TFA in the skins of “Niagara Rosada”, “Hasang” and “Changbaijiu” were correlated to their extremely high contents of procyanidin dimmer levels. For monomers, catechin and epicatechin were the most common, but their levels were still low in most non-muscadine grapes.There were 8 flavan-3-ol compounds detected in all, including 4 monomers , 3 dimers and 1 trimer . In the V. xunyangensis “Mi”, the V. amurensis × V. vinifera hybrid “Zuoyouhong” and V. dividii “Black pearl” . In addition, V. quinquangularis “Mao”, V. labrusca “Fredonia” and V. amurensis “Changbaijiu” also had a high TCA content . However, only trace cinnamic acids were detected in V. vinifera “Merlot” and “Cabernet Sauvignon”. Nine different cinnamic acids were found in the skins among the cultivars investigated, with chlorgenic acid, hexose ester of ferulic acid, and fertaric acid were more common than others. Fertaric acid was the most abundant cinnamic acids in the East Asia group, accounting for 49.87% of TCAs  contents in the tested samples ranged from trace amounts to 230 μg caffeic acid equivalence (CAE)/g DW . The thr of TCAs .V. ficifolia “Sangye”, whereas the lowest content was found in V. labrusca “Catawba” (0.58 μg GAE/g DW).Five benzoic acids were also identified, and hexose ester of vanillic acid was the most common, followed by hexose ester of chlorgenic acid . In geneEllagic acids were uniquely found in muscadine grapes. There were 647.68 and 525.16 μg ellagic acid equivalence (EAE)/g DW of total ellagic acids (TEAs) in “Noble” and “Alachua”, respectively . Hydrolytrans-pecied), were identified in all the tested grape berry skins (V. Vinifera “Merlot” skins (167.06 μg RE/g DW), followed by V. ficifolia “Sangye” and V. rotundifolia “Noble” . There were trace or no stilbenes found in American grape cultivars and Euro-American hybrids, and also in most V. amurensis cultivars and hybrids, except “Shuangyou”, “Zuohongyi” and “Hasang”  were subjected to Principle Component Analysis (PCA) in order to separate these grapes according to their skin phenolic characteristics. The first three principal components (PCs) possessed relatively high percentages of total variance. PC1 described 27.94% of the variance, which had high contributions from 4′-substituted anthocyanins, quercetin derivatives, myricetin derivatives, total flavonols, and monomeric and total flavon-3-ols, as well as total ellagic acids. PC2 described 25.32% of the variance, and correlated with 3′,4′-substituted, 3′,4′,5′-substituted, methylated, non-methylated, diglucosides, total anthocyanins and total benzoic acids. PC3 accounted for 12.50% of the variance and was mostly described by kaempferol derivatives, and polymeric and total flavon-3-ols.V. Vinifera, North American grape cultivars, Euro-American hybrids, and the V. amurensis × V. vinifera hybrid “Hasang”. This group was linked by their relatively high contents of kaempferol derivatives or polymeric or total flavon-3-ols in the skins. Group B, in the negative PC3 axis, was comprised of all the East Asian grapes and Euro-Asian hybrids except “Hasang”. This group was matched through no or low content of kaempferol derivatives or polymeric or total flavon-3-ols in their skins. The two mascadine grapes comprised group C, located in the significantly higher position of the PC1 axis. This group was characterized by the presence of pg-3,5-diglucosides and ellagic acids, and by high contents of monomeric flavan-3-ols and flavonols.V. amurensis grape cultivars, three V. amurensis × V. vinifera hybrids, four American cultivars and three Euro-American hybrids were collected from the experimental vineyard of the grape germplasm repository of China Agricultural University in Beijing. The two V. vinifera grape cultivars were from the nearby vineyard of Ji county in Tianjing. The berries of the other four East Asian species were collected from the experimental vineyard of the grape germplasm repository of Henan Academy of Agricultural Science in Zhengzhou. The berries of the two muscadine grape cultivars were collected from the experimental vineyard of Guangxi Academy of Agricultural Science in Nanning . Dried specimens were ground thoroughly with a stainless-steel grinder , and stored in vacuum-packaged polyethylene pouches at −20 °C for subsequent analysis.O-glucoside, quercetin, catechin and ellagic acid, were all obtained from Sigma-Aldrich . The HPLC grade reagents, methanol, formic acid, acetic acid and acetonitrile, were purchased from Fisher Scientific Co. . Folin-Ciocalteu phenol reagent (2 N) and 2,4,6-tripyridyl-s-triazine (TPTZ) (≥99%) were purchased from Sigma-Aldrich . 6-Hydroxy-2,5,7,8-tetramethylchro-man-2-carboxylic acid (Trolox) (≥99%) was purchased from Alexis . All other analytical grade chemicals and reagents were purchased from Lanyi Co. .The standards, caffeic acid, gallic aid, resveratrol, malvidin-3-A protocol developed in our lab was used for the total phenol extraction and anthocyanin analysis . BrieflyFor the analysis of non-anthocyanin phenolics including phenolic acids, stilbenes, flavonols, flavan-3-ols and ellagic acids, the extraction was performed according to Jin, He, Bi, Cui and Duan (2009) . Briefly−1 and the column temperature was set at 50 °C. The injection volume was 30 μL and the detection wavelength was 525 nm. MS analyses was used Electrospray ionisation (ESI), positive ion model, 35 psi nebulizer pressure, 10 mL min-1 dry gas flow rate, 350 °C dry gas temperature, and 100–1000 m/z scan range [Anthocyanin analysis were performed on an Agilent 1100 series LC-MSD trap VL equipped with a G1379A Degasser, a G1311A QuatPump, a G1313A ALS, a G1316A Column, a G1315B DAD and a Kromasil-C18 column . The mobile phase was: aqueous 2% formic acid as solvent A, and acetonitrile containing 2% formic acid as solvent B. The gradient profile was from 6% to 10% B for 4 min, from 10% to 25% B for 8 min, isocratic 25% B for 1 min, from 25% to 40% for 7 min, from 40% to 60% for 15 min, from 60% to 100% for 5 min, and from 100% to 6% for 5 min. The flow rate was 1.0 mL·minan range .−1 and the column temperature was set at 25 °C. The injection volume was 2 μL and the detection wavelength was 280 nm. MS analyses were used as follows: Electrospray ionisation (ESI) interface, negative ion model, 35 psi nebulizer pressure, 10 mL·min-1 dry gas flow rate, 325 °C dry gas temperature, and 100–1000 m/z scan range [Analysis of non-anthocyanins were performed on an Agilent 1200 series equipped with a G1322A Degasser, a G1312B Bin pump, a G1367C HiP-ALS, a G1316B TCC, a G1314C VWD and a ZORBAX SB-C18 column . The mobile phase was: water solution with 1% acetic acid as solvent A, and acetonitrile solution with 1% acetic acid as solvent B. The gradient profile was from 5% to 8% solvent B for 10 min, from 8% to 10% B for 8 min, from 10% to 15% B for 22 min, from 15% to 20% B for 10 min, from 20% to 30% B for 3 min, from 30% to 50% B for 5 min, from 50% to 100% B for 4 min, and isocratic 100% B for 4 min. The flow rate was 1.0 mL·minEach group of phenolic compounds was quantified using its representative compound as standard. Anthocyanins, flavonol, flavan-3-ols, cinnamic acids, benzoic acid, stilbenes and ellagic acids were respectively expressed as micrograms of Malvidin-3-glucoside (MGE), quercetin equivalence (QE), catechin equivalence (CE), caffeic acid equivalence (CAE), gallic acid equivalence (GAE), resveratrol equivalence (RE) and ellagic acid equivalence (EAE)/g of dry matter (DM).All analyses were expressed as means ± standard deviations (S.D.) of duplicate. All data were subjected to one-way analysis of variance (ANOVA) using SPSS 16.0 (SPSS Inc.) at the 95% confidence level. The principal component analysis (PCA) was performed to investigate skin phenolic-based species relationships and correlate phenolic compositions to species/cultivars.V. vinifera grapes and V. rotundifolia grapes, respectively, while both existed in other species and hybrids. In the grapes originating from Eurasia, 3′,4′,5′-substituted and methylated anthocyanins were dominant, while the proportions of total anthocyanins were relatively low in grapes originating from North America. In addition, acetyl anthocyanins were more abundant in V. vinifera grapes than other species grapes. Furthermore, quercetin-3-glucuronide and quercetin-3-glucoside were common in the skins of Euvitis species but were not detected in muscadine grapes. The muscadine grape skins possessed more non-anthocyanin phenolics than other grapes due to their richness in flavonols, flavan-3-ol monomers and ellagic acids. Flavan-3-ols were generally sparse in the skins of the East Asian grapes and hybrids. According to PCA, all the samples could be divided into three groups: V. rotundifolia grapes, East Asian grapes and hybrids, and other grapes.Skin phenolic compositions varied widely among grape cultivars with different genetic backgrounds and originations. Monoglucoside and diglucoside anthocyanins were only detected in


Levels of genetic diversity can strongly influence the dynamics and evolutionary changes of natural populations. Survival and disease resistance have been linked to levels of genetic diversity in eusocial insects, yet these relationships remain untested in gregarious insects where disease transmission can be high and selection for resistance is likely to be strong.Malacosoma californicum pluviale from four different island populations to determine the relationship of genetic variability to survival and disease resistance. In addition these genetic markers were used to elucidate the population structure of western tent caterpillars. Multiple paternity was revealed by microsatellite markers, with the number of sires estimated to range from one to three per family (mean ± SE  = 1.92±0.23). Observed heterozygosity (HO) of families was not associated to the resistance of families to a nucleopolyhedrovirus (NPV) , a major cause of mortality in high-density populations, but was positively associated with larval survival . Genetic differentiation among the families was high , and families from the same island were as differentiated as were families from other islands.Here we use 8 microsatellite loci to examine genetic variation in 12 families of western tent caterpillars, We have been able to describe and characterize 8 microsatellite loci, which demonstrate patterns of variation within and between families of western tent caterpillars. We have discovered an association between larval survival and family-level heterozygosity that may be relevant to the population dynamics of this cyclic forest lepidopteran, and this will be the topic of future work. For gregarious or colonial animals the opportunities for disease transmission among individuals are likely to be high and thus, selection for resistance to disease is also expected to increase Daphnia magnaPotamopyrgus antipodarumCoenagrion puellaMalacosoma californicum pluviale to infection by a nucleopolyhedrovirus (NPV) that occurs regularly at high host population densities Tests of disease resistance in field populations of animals, particularly invertebrates, is challenging and data are limited, although detailed studies have been carried out on M. americanum) investigation of allozyme variation revealed that multiple mates were common Given the extensive work on polyandry, genetic variation and potential disease resistance in colonial insects th instar of development, (4) the extent of polyandry among families and (5) the association of genetic variability to the density of populations in the field. This study of the neutral genetic variation among families of tent caterpillars from different island populations can also be used to assess the levels of geographic subdivision that occur among families from the four islands surveyed.Genetic studies that evaluate hypotheses to explain population fluctuations in forest Lepidoptera remain underrepresented in the literature. Microsatellite markers allow for the evaluation of levels of neutral variation, with greater resolution than allozymes, and we have developed eight loci for western tent caterpillars to explore associations between genetic variation and fitness. We have used these microsatellite variants to determine (1) levels of genetic variability within and among families, (2) the relationship between genetic variability and the resistance of the families to nucleopolyhedroviral infection, (3) the relationship between genetic variability and the larval survival to the 4Here we describe microsatellite variants that we have used to explore the potential relationships between genetic variability (observed levels of heterozygosity) and fitness characteristics of the western tent caterpillar. We test the hypothesis that increased heterozygosity is associated with greater resistance of families to nucleopolyhedroviral infection and to better survival. Contrary to our predictions, disease resistance was not related to genetic heterozygosity at the family level. Larval survival, however, was positively correlated with observed heterozygosity.Specific permits were not required for field collections of egg masses and permission was not required to obtain collections at all field sites. Because tent caterpillars are considered to be minor pests the public does not value them and removal of egg masses is in fact valued. They have neither endangered nor protected status. Collection sites are roadsides and “waste” areas.Alnus rubra, and wild cherry, Prunus emarginata, Saturna Island  from red alder, Mandarte Island  on the Haro Straight east of southern Vancouver Island from wild rose, Rosa nutka, and Westham Island  in the delta of the Fraser River, south of Vancouver from crab apple, Malus diversifolia and hawthorn, Crataegus monogyna  from red alder, monogyna . These pmonogyna  [20]. Egth instar larvae in each family were challenged with M. c. pluviale NPV (McplNPV) in bioassays that have been reported elsewhere 50) were determined using generalized linear models with a binomial distribution and a logit link structure . There was no interaction effect with dose, thus the dose-response lines were parallel, enabling LD50s to be compared across families and sites The remaining 4E. coli strain DH5α  by electroporation. Colonies were enriched for four distinct oligonucleotide probes . Ninety-nine colonies were sequenced and of these 83 contained microsatellites (GenBank Accession No. JN861644 – JN861726). Primers were designed for a subset of the microsatellites using DesignPCR version 1.03 (Research Genetics Inc.). Twenty-four microsatellites were tested for polymorphism using library DNA and seven additional individuals. Polymerase chain reactions (PCR) for all microsatellites used 2 ng of genomic DNA, 0.2 mM dNTP, (1X) PCR buffer, 0.025 units of Bio Taq, 2 mM MgCl2, 0.6 μmol each primer, (1X) sucrose/cresol red, and 5.15 μL of H2O in a total reaction volume of 10 μL. The PCR conditions were as follows: 94°C for 3 min, cycled 35 times at 94°C for 40 sec, 55 to 57°C for 40 sec, 72°C for 30 sec, with a final extension of 72°C for 4 min. Products were run on a 3% agarose gel and reviewed for polymorphism.A repeat enriched library was constructed at the Genetic Identification Service  from a single caterpillar. Caterpillar DNA was isolated using DNeasy Blood and Tissue® kit . Digested DNA (350–700 bp fragments) was incorporated into 2  from 10 caterpillars from each of the 12 families. One individual from each of eight tent caterpillar families were used to further screen 21 of the microsatellites for polymorphism at the Genetic Data Centre . Thirteen of the 21 microsatellites had low polymorphism  and were not investigated further. The other eight microsatellites showed a high level of polymorphism and therefore were selected for the investigation of the 12 tent caterpillar families, which were arbitrarily selected from a total of 30 families  with a minimum of four size standards (50–350 bp or 50–700 bp LICOR Inc.) per 64 well gel and two to four reference samples. Gels were scored using SAGA 2.0 for microsatellites (LICOR). The accuracy of marker information was verified by repeating PCR and retyping samples within families for markers where multiple paternity was inferred (∼20% of samples verified).2, 3 pmol forward and reverse primers , 6.25 μL H20 for a total reaction volume of 10 μL. PCR conditions were as follows: 95°C for 3 min, cycled 35 times at 94°C for 40 sec, 56°C for 40 sec, 72°C for 30 sec, with a final extension of 72°C for 4 min. Four, 10 μL reactions were performed for each individual sequenced and amplified products were pooled prior to purification with a sodium acetate precipitation, and sent to Eurofins MWG Operon  for sequencing. Sequences were aligned using Bioedit Sequence Alignment Editor Sequences were obtained from four individuals at locus A106 to determine the cause of the large range of allele sizes (290–470 bp). Two of the individuals  sequenced were homozygous for alleles 310 bp in size and the other two  were homozygous for alleles 470 bp in size. PCR reactions used 10 ng of genomic DNA, 0.0625 mM dNTP, (1X) PCR buffer , 0.25 units of Taq (Bio Basic Inc.), 2 mM MgClTo test for a step-wise mutation model the frequency distribution for each locus was constructed and normality was assessed visually and tested statistically using the Shapiro-Wilk normality test a), number of effective alleles (Ne), allele frequency range, expected heterozygosity (HE) O) for each locus and family group. Mean rarefied allelic richness (RT) was calculated for each loci and family in FSTAT 2.9.3.2 50 for NPV exposure, and survival in JMP 8.0.2. We used population size estimates from 2002, since the egg masses collected were the result of matings in 2002. Population size and LD50 were log transformed and survival was arcsine square root transformed to ensure that the assumption of normality was met. We performed post-hoc power analyses in SAS version 9.3 (SAS Institute Inc. 2010) to determine the statistical power for each correlation examined and determined the sample size that would have been required to achieve 80% power.GenoDive version 2.0b20 ST values were estimated in Arlequin version 3.11 ST values were tested by performing 10 000 random permutations and the Bonferroni correction was used to account for multiple comparisons. Analysis of molecular variance (AMOVA) was performed in Arlequin version 3.11 to test for hierarchical genetic structure among families from the four different islands and significance was assessed by performing 10 000 random permutations of the data.Global and pairwise Ff, also referred to as FIS, was used to test for Hardy-Weinberg equilibrium (HWE) for each family-locus combination and overall for families and loci The inbreeding coefficient T), disease resistance, and population size in 2002 (log transformed) (JMP 8.0.2). Allelic richness was used instead of observed heterozygosity because it is more sensitive to changes in the number of alleles. Post-hoc power analyses were performed in SAS 9.3 to determine the statistical power of each correlation and the sample size required to achieve 80% power.The minimum number of fathers was reconstructed for each family with unknown parental genotypes using an exhaustive search algorithm based on Mendelian segregation and population genotypic frequencies in GERUD 2.0 Microsatellites were identified in 83 of the 99 cloned colonies and contained di, tri, and tetra nucleotide repeats. Primers could not be designed for 23 of the microsatellites due to sequence duplication and unsuitable flanking regions for primer design. Twenty-one microsatellites were tested for polymorphism and of those, eight di-nucleotide loci were selected based on polymorphism for further analysis. BLASTn results produced no significant matches to coding regions for these eight microsatellites.P<0.0001) and rather shows a bi-modal distribution.A step-wise mutation model may be inferred for microsatellite markers if their size frequency distribution is normally distributed. The Shapiro-Wilk normality test indicated that one of the eight loci (A106) we examined was not normally distributed  . A compaa) ranged from 4 to 18 and rarefied allelic richness (RT) varied from 3.0 to 8.1 for each locus (E) and observed heterozygosity (HO) ranged from 0.35 to 0.74 and 0.24 to 0.91, respectively. Observed heterozygosity was higher than the expected heterozygosity for all but two (B108 and B121) of the eight loci examined. None of the loci, however, showed significant departures from Hardy-Weinberg equilibrium . In addition, there was no evidence of linkage disequilibrium between any pairs of loci (P>0.05).The number of observed alleles . We also predicted that multiple paternity would be more prevalent in families at sites with the highest population size. However, there was no relation between the minimum number of sires and population size in 2002 . In addition, the association between the number of sires and disease resistance was weakly, but non-significantly positive . The statistical power of the correlations between the number of sires and population size in 2002 and disease resistance were relatively low and would require at least an additional 60 samples to potentially generate a significant result. However, it is difficult to obtain large data sets for gregarious insects because only one value can be used per family and therefore the required sample size represents the number of families and not individuals.Families with multiple sires were expected to have greater genetic diversity than those with a single sire. In agreement, the number of sires per family was positively correlated with allelic richness  and allelic richness (RT) were greatest for families from Saturna Island, which had the highest population density in 2002 when egg masses were collected and reached peak density in 2003; a year prior to populations from other islands. The mean effective number of alleles and allelic richness were lowest on Mandarte Island, where the population is more isolated and reached peak size in 2004 .Genetic diversity measures for 12 families from four island locations are described in  in 2004 . The expO: r = 0.067, F1,12  = 0.045, P = 0.835). Statistical power, however, was low (0.07) for this test and would have required in excess of 1000 families sampled to potentially generate a significant result. Within families, observed heterozygosity levels were similar and there was no evidence of significant departures from Hardy-Weinberg equilibrium .We predicted that the high density of tent caterpillars in 2002, prior to the peak in population size would be positively associated with observed heterozygosity due to the increased availability of mates, however no relationship was identified . The power of this test was low (0.128) and would have required samples from at least 230 more families. We also predicted that larval survival would be positively correlated with measures of genetic diversity. In agreement with this, we observed a positive multi-locus correlation for families between mean observed heterozygosity and survival from egg hatch to the 4th instar of development . With the exception of two families from Galiano Island , all pairwise comparisons tested showed significant differentiation between families (P<0.05). High levels of differentiation were often observed between families from the same island . Consistent with these results, differentiation among families from the same island contributed significantly . In addition, differences among families from different islands accounted for the greatest amount of variation  and there was no significant differentiation among islands .The global Ffamilies ; P<0.05.M. c. pluviale, we found that multiple paternity was common among families from four island populations. However, we found no relationship between viral resistance, as measured by the LD50, and the average observed heterozygosity of families. The average heterozygosity of families was, however, positively associated with larval survival. This finding is in agreement with other studies that have identified a positive association between genetic diversity and fitness benefits in eusocial insect species Using eight new microsatellites, developed specifically for Malacosoma c. pluviale is primitively social and thus, individuals in families work together to build tents and mark food trails with chemical cues to attract siblings. But tent caterpillars lack complex social interactions such as grooming, food storage and provisioning behaviours that are characteristic of eusocial species 50 and number of fathers represented in a family was largely determined by two families with particularly high resistance, one with two fathers and one with three.Malacosoma americanum, identified, using allozyme variation, that at least 19% of females were multiply mated M. c. pluviale sires per family ranged from one to three, with an average of two fathers per family. In contrast to the results for M. americanumM. c. pluviale sires made a substantial genetic contribution to the offspring of egg clutches from multiply mated females. Our results are in line with those observed for other invertebrate species with internal fertilization Polyandry is prevalent in many lepidopteran species M. c. pluviale since its populations fluctuate and are subject to environmental perturbations such as changes in temperature, weather, and food availability The benefits of polyandry must outweigh the costs for it to be maintained Bombus terrestris colonies where worker heterogeneity was manipulated by artificially inseminating queens, increased worker heterozygosity was associated with reduced infection to the common parasite, Crithidia bombi (Trypanosomatidae) A large body of research has focused on the benefits of multiple mating in relation to disease resistance in eusocial insects Highly variable codominant microsatellite markers are well suited to this type of study of paternity and population structure Malacosoma c. pluviale populations go through boom and bust dynamics. The population trough that occurred in 1999–2000 may have increased the likelihood of strong linkage disequilibria Multi-locus heterozygosity fitness correlations that describe the association between individual heterozygosity and fitness related traits have been well documented in natural populations Zeiraphera diniana in western Europe, FST values were higher (FST = 0.065) between populations feeding on two different host tree species than were the values among populations on the same host species (FST = 0.026 and 0.002) Panolis flammea in England and Scotland were surveyed using four electrophoretic loci and significant variation existed among populations (FST = 0.109). The genetic similarity among populations of western tent caterpillars from different islands contrasts with these studies. Sample sizes in our study are small and only one year at high density has been studied.An additional finding of this study is that the greatest amount of genetic variation we observed was among families and this variation among families was similar within and among different islands. Genetic differentiation among populations of other cyclic forest Lepidoptera has been investigated using electrophoretic methods. For larch budmoth, M. disstria, which periodically demonstrates population outbreaks across North America The microsatellite variation described here can be used in future studies to determine if genetic variation and/or the number of mates varies with population density and if island populations are more differentiated at low population densities. The level of polyandry found here is surprising and could be under positive selection or could be the result of very high density and forced copulations. More behavioural work is required. Finally, it will be interesting to determine if these genetic markers can be used for other species of tent caterpillar such as the eastern tent caterpillar or the forest tent caterpillar, Table S1Malacosoma californicum pluviale families collected from Galiano, Mandarte, Saturna, and Westham Island on the west coast of British Columbia, Canada.Intercorrelations between genetic diversity measures. Intercorrelations between genetic diversity measures for the 12 families of (PDF)Click here for additional data file.


Hemidactylus mabouia in Florida using 12 microsatellite loci. We investigated dispersal patterns at the regional scale (metropolitan areas), statewide scale (state of Florida), and global scale (including samples from the native range). Dispersal was limited at the smallest, regional scale, within metropolitan areas, as reflected by the presence of genetic structure at this scale, which is in agreement with a previous study in this same invasion at even smaller spatial scales. Surprisingly, there was no detectable genetic structure at the intermediate statewide scale, which suggests dispersal is not limited across the state of Florida. There was evidence of genetic differentiation between Florida and other areas where H. mabouia occurs, so we concluded that at the largest scale, dispersal was limited. Humans likely contributed to patterns of dispersal at all three scales but in different ways. Infrequent low-volume dispersal has occurred within regions, frequent high-volume dispersal has occurred across the state, and infrequent long-distance dispersal has occurred among continents at the global scale. This study highlights the importance of considering different modes of dispersal at multiple spatial scales to understand the dynamics of invasion and range expansion.Dispersal influences both the ecological and evolutionary dynamics of range expansion. While some studies have demonstrated a role for human-mediated dispersal during invasion, the genetic effects of such dispersal remain to be understood, particularly in terrestrial range expansions. In this study, we investigated multimodal dispersal during the range expansion of the invasive gecko  Dispersal plays a critical role in determining the ecological and evolutionary dynamics of range expansion. Natural dispersal in terrestrial habitats has typically been thought to be quite limited and to occur primarily over short distances, giving rise to relatively small founding populations with limited genetic diversity and slow rates of spread . Human-mHuman-mediated dispersal can have important demographic and genetic effects, sometimes resulting in high propagule pressure and genetic diversity at the introduction stage of invasion . IncreasWhere multiple modes of dispersal are occurring simultaneously, they are most likely to be detected by investigation of dispersal at multiple spatial scales. Molecular genetic analyses using microsatellites have made it possible to reconstruct dispersal patterns at multiple scales during range expansion . SeveralHemidactylus mabouia , and communal nesting  in Genetic Data Analysis  or five populations in Miami , although the relationship disappeared with removal of the Avon Elementary population (P= 0.41), suggesting that this population may have been largely responsible for the correlation.There was no evidence for isolation by distance among four populations in St. Petersburg/Tampa (FST (θ) values among populations within regions ranged from 0 to 0.17 ( to 0.17 . In St. K= 4) among the five populations. In Sebring, there were two genetic clusters but they did not clearly correspond to sample sites; most sample sites were not strongly assigned to a particular cluster. In Miami, there was only one genetic cluster, indicating relative panmixia at the regional scale  was attributable to variation within regions. This seemingly counterintuitive result suggested that there was greater structure at a smaller geographic scale than across the state, and was explored further through analyses at the statewide scale.An AMOVA on all three regions within Florida revealed that while 0% of the variation was attributable to differentiation among regions, 8%  or when populations within regions were pooled  or the West coast .Across the state of Florida, expected heterozygosity ranged from 0.42 to 0.59, and allelic richness ranged from 2.27 to 3.19 after rarefaction to four individuals. There was evidence for heterozygote deficit in a few populations , which lFST (θ) value for all the populations considered in both transects was 0.06. The overall FST (θ) for the West coast transect was 0.11, and 0.07 when populations in the same region were pooled. On the East coast, the overall FST (θ) was 0.04, and 0.03 when regions were pooled. Pairwise θ values among individual populations on either coast ranged from 0 to 0.26 when populations were considered independently and from 0.004 to 0.26 when populations were pooled within regions.The overall K= 2, the S. America/Caribbean samples were clustered with those from Africa, and the Florida samples formed their own cluster. Allelic richness after rarefaction to three individuals (due to few data in the African samples) was significantly different among all three continents, with highest values in Africa, lowest values in South America/Caribbean, and intermediate values in Florida (Africa–S. America/Caribbean Z=–34.0; P= 0.005; Africa–Florida Z=–28.0; P= 0.027; S. America/Caribbean–Florida Z= 28.0; P= 0.027; Bayesian population structure analysis revealed the presence of three distinct clusters, with each continent comprising an independent cluster . When K=H. mabouia occurs, and this suggests that ongoing dispersal of H. mabouia among continents is rare. Together, these results underscore the role of human-mediated dispersal during terrestrial invasion and also demonstrate the importance of investigating patterns at multiple spatial scales during invasion and range expansion.We tested for genetic structure at three spatial scales, and found different patterns at each scale, which supports our hypothesis of multimodal dispersal . The preH. mabouia for around 20 years. In a separate study, we demonstrate that population structure arising from colonization is ephemeral, and that over time, gene flow among sites gradually erodes the signature of colonization (Significant population structure was present at the regional scale among sites within St. Petersburg and Sebring, although in Sebring, the inferred clusters did not correspond to sample sites. This may reflect recent and ongoing gene flow among sites near Sebring. The lack of genetic structure among sites in Miami is likely attributable to the fact that the Miami area has been colonized by nization . When reThe presence of population structure at the regional scale would appear to contradict the lack of structure at the statewide scale without consideration of multiple dispersal modes. With limited natural dispersal, we would expect to find population structure at both spatial scales. Because we found population structure at the regional scale but not at the larger statewide scale, we conclude that the colonization process must involve different dispersal modes at different spatial scales. In other studies of multimodal dispersal involving natural short-distance dispersal and some form of long-distance dispersal, the long-distance component is generally attributed to either natural long-distance dispersal associated with a particular life stage (especially in aquatic organisms; Even human-mediated dispersal in terrestrial habitats may take different forms at different scales. Dispersal within regions is likely a combination of natural dispersal and low-volume human-mediated dispersal through intentional translocations, localized shipments, or movements on recreational vehicles. However, human-mediated dispersal at the statewide scale is more likely to be a form of mass dispersal through the shipment of larger quantities of goods and containers between hotspot distribution points. Nevertheless, the sharp contrast between the restricted gene flow at the regional scale and the frequent gene flow at the statewide scale suggests that different dispersal modes are prevalent at different spatial scales. In spite of evidence for human-mediated dispersal during the introduction stage of invasion, evidence for this phenomenon during range expansion remains scarce, especially in terrestrial systems. While a few studies have explicitly investigated genetic patterns at multiple scales , ours isH. mabouia colonized the New World, and there is not a significant amount of ongoing gene flow among continents. Therefore, we conclude that transoceanic dispersal in H. mabouia is somewhat limited, and does not play a major role in shaping genetic patterns within Florida.Many studies have focused on gene flow from the native range as a primary factor in determining the genetic patterns associated with invasion . Some stEvidence for scale-dependent human-mediated dispersal has important implications for biological invasions and urban ecology, and these implications extend to a variety of other organisms. The role of human-mediated dispersal in other organisms likely depends to some extent on traits of the organisms. For example, both sociality and commensalism with humans may increase the numbers of individuals involved in human-mediated dispersal events. House geckos are known to share shelters and egg-laying sites on buildings , and thiMass dispersal may have consequences for the ecology and evolution of invasive populations . We foun