Ifremeria nautilei.Deep-sea hydrothermal vents provide patchy, ephemeral habitats for specialized communities of animals that depend on chemoautotrophic primary production. Unlike eastern Pacific hydrothermal vents, where population structure has been studied at large (thousands of kilometres) and small (hundreds of meters) spatial scales, population structure of western Pacific vents has received limited attention. This study addresses the scale at which genetic differentiation occurs among populations of a western Pacific vent-restricted gastropod, Ifremeria nautilei separated by a few meters within a single vent field to distances as great as several thousand kilometres between back-arc basins that encompass the known range of the species. No genetic subdivisions were detected among patches, mounds, or sites within Manus Basin. Although I. nautilei from Lau and North Fiji Basins (~1000 km apart) also exhibited no evidence for genetic subdivision, these populations were genetically distinct from the Manus Basin population.We used mitochondrial and DNA microsatellite markers to infer patterns of gene flow and population subdivision. A nested sampling strategy was employed to compare genetic diversity in discrete patches of Ifremeria nautilei from widespread populations that occupy the North Fiji and Lau Basins. A robust understanding of the genetic structure of hydrothermal vent populations at multiple spatial scales defines natural conservation units and can help minimize loss of genetic diversity in situations where human activities are proposed and managed.An unknown process that restricts contemporary gene flow isolates the Manus Basin population of  The spatial scales at which individuals within a population interact and the geographic extent of larval dispersal shape the dynamics of marine populations. Dispersal capabilities of some species extend across entire ocean basins , but larSpecies dependent on deep-sea hydrothermal vents are restricted to patchy, ephemeral habitats that limit the areal extent and occurrence of populations. Hydrothermal vent fields are found on mid-ocean ridges, back-arc spreading centres, and submarine volcanoes . OrganisRimicaris exoculata on the Mid-Atlantic Ridge [At mid-ocean ridges, deep-sea hydrothermal vents are distributed along roughly linear axes that may function as dispersal corridors -17. Geogic Ridge -20) but Ridgeia piscesae [Evidence for isolation-by-distance in vent species has sometimes been ambiguous due to small sample sizes and inconsistency in the resolution of various genetic markers . Considepiscesae . Life hipiscesae .Riftia pachyptila, separated by as little as 400 m in a venting area along the East Pacific Rise, although sample sizes were small . More cdistance  at greatdistance ,29.In contrast to mid-ocean ridge systems, limited attention has been afforded to the population structure of vent organisms from western Pacific back-arc basins. These basins are distributed in a non-linear pattern, reflecting the complex tectonic history of the region . HydrothBathymodiolus brevior [Alviniconcha represent a cryptic species complex composed of at least three evolutionary lineages, one that occurs at hydrothermal vents in North Fiji Basin, one that is restricted to vents in the Marianna Trough, and one that co-occurs in both Manus and North Fiji Basin [Because back-arc basin hydrothermal systems in the western Pacific are located on isolated ridge segments , it has been hypothesized that reduced connectivity among western Pacific back-arc basins may yield more endemic vent fauna within discrete back-arc basins . Some sp brevior ), wherea brevior ). Provanji Basin . A similIfremeria nautilei is a provannid gastropod that occurs in Manus, North Fiji, and Lau Basins and depends on sulphur-oxidizing bacterial endosymbionts for nutrition. Sessile adults live in discrete patches near the effluent of diffuse-flow hydrothermal vents [I. nautilei exhibits distinct mitochondrial haplotypes in Manus and North Fiji Basins [sites), among sulphide mounds within vent fields (henceforth mounds), or among discrete patches on vent mounds (henceforth patches).al vents ,30. Femaal vents . Prelimii Basins , but popIfremeria nautilei from hydrothermal vents in Manus, North Fiji, and Lau Basins at multiple scales, ranging from meters to thousands of kilometres  and genetic differentiation. Genetic markers for differentiation at these scales included partial sequences of mitochondrial cytochrome-c-oxidase subunit I, and an array of nuclear DNA microsatellite loci [COI sequence data and dependent on mutation rates, from ecologic processes, revealed by microsatellite allele frequencies and based on the recombination of alleles with each generation [Ifremeria nautilei are adapted for long-distance dispersal [I. nautilei disperse in a manner consistent with other sessile invertebrates with specialized habitat needs [We examined genetic population structure of s Figure . A nesteite loci . By compispersal , populatat needs  genetic Ifremeria nautilei were sampled from Manus, North Fiji, and Lau Basins  were identified from 158 Ifremeria nautilei sampled from Manus Basin; an additional 25 haplotypes were identified from 117 individuals from North Fiji and Lau Basins. Haplotype diversity (dH) ranged from 0.59 to 0.95  [e sweep) , with twRs) did not vary significantly among patches, mounds, or sites  were monomorphic at the patch level but were polymorphic among patches.Nine microsatellite loci were amplified from 40 to 66 individuals per site within Manus Basin  failed to amplify in any North Fiji or Lau Basin samples. The total number of alleles per locus ranged from 2 to 23 (mean = 6.2). In permutation tests, allelic richness (Rs) did not vary significantly among sites or basins , but one microsatellite locus (Ifr043) was under positive selection  at the basin level.Two microsatellite loci deviated significantly from Hardy-Weinberg Equilibrium at sites within North Fiji Basin (IDP) and probability of sibling identity tests (SIBP) indicated that the nine microsatellite markers identify individuals (ID P= 1.5 × 10-6), including those that shared 50% genetic similarity (SIB P= 3.5 × 10-3). IMa coalescent models require that microsatellite markers adhere to the stepwise mutation model; only four of nine microsatellite markers  adhered to this model and could be used for IMa analysis. Identity tests for these four markers suggested that they are insufficient for assessment of population structure .Identity tests were used to assess the utility of each microsatellite marker set. Within Manus Basin, probability of identity tests  failed to amplify, were out of equilibrium, or were under selection. These markers were excluded from all analyses involving North Fiji and Lau Basins. The five remaining microsatellite loci could identify individuals (ID P= 4.0 × 10-4), even those that share 50% genetic similarity (SIB P= 4.4 × 10-2). Only two of those microsatellites (Ifr052 and Ifr078) adhered to the stepwise mutation model and could be used in IMa analyses. Identity tests for these two markers suggested that they are insufficient for assessment of population structure .Within North Fiji and Lau Basins, four of nine microsatellite markers (n = 10), followed by Solwara 1 (n = 8) and Solwara 8 (n = 7).Mitochondrial genealogies revealed two frequent haplotypes at all Manus Basin sites ; pairwise comparisons of ST Fand ST φrevealed no significant genetic differentiation (P > 0.05); hierarchical analysis of ST Fand ST φdid not reveal any significant population differentiation among nested samples  indicated no variation among haplotypes using either mitochondrial or microsatellite markers  could not rule out the possibility of equal and bidirectional migration within Manus Basin populations. Due to the high level of connectivity between Manus Basin samples, it is unlikely that estimates of gene flow would converge on the most likely solution. The posterior probabilities display multiple peaks and gradually increasing probabilities and thus results from these analyses should be approached with caution.As reported above, the few microsatellites that adhered to the stepwise mutation model were insufficient to adequately assess gene flow. Only the mitochondrial I. nautilei from all three western Pacific basins, coalescent estimates of migration rate (IMa) suggest that migration between North Fiji and Lau Basin is high  had a sufficient sample size to test and reject the hypothesis that self-recruiting patches of I. nautilei might create the appearance of panmixia within this sample set.Ifremeria nautilei from South Su had the highest abundance of private haplotypes and private alleles. Under a scenario of colonization with subsequent migration, this pattern could suggest that South Su might serve as a source population that contributes individuals to other sites sampled in Manus Basin. This directional gene flow is consistent with the path of the St. George's Undercurrent, which enters Manus Basin from the southeast and travels northwest, encountering South Su first, then Solwara 1 and Solwara 8, before merging with the Vitiaz Straight Undercurrent to form the New Guinea Coastal Undercurrent [Ifremeria nautilei. Rapid population expansion, as suggested by Fu's S Fand the star-like mitochondrial genealogies, could account for the emergence of private haplotypes and alleles at each sampled site within Manus Basin. Alternatively, the negative Fu's S Fvalues could be a result of a selective sweep on the mitochondrial genome. Additional sequenced-based nuclear markers would be needed to rule out this possibility.rcurrent . This hyIfremeria nautilei sampled from across the known range of the species in the south western Pacific could be subdivided genetically into two populations, one restricted to Manus Basin and one distributed throughout North Fiji and Lau Basins. Under an isolation-by-distance scenario, genetic differentiation is expected to gradually increase with distance [I. nautilei populations between Manus Basin and North Fiji/Lau Basins may have existed, with occasional migration, for several hundred thousand generations  that form effective dispersal barriers for a wide range of taxa . A relac basins  and is cc basins . The obsIfremeria nautilei is not likely the result of a colonization event. Colonization would result in a founder effect, where the founded population contains a subset of alleles from the source population [The phylogeographic break between Manus and North Fiji/Lau populations of pulation . The sinpulation . HaplotyIfremeria nautilei occurs throughout Manus, North Fiji, and Lau Basins, populations of I. nautilei follow the trend of greater endemism and limited connectivity hypothesized for species endemic to back-arc basin spreading centres [I. nautilei is distributed through North Fiji and Lau Basin, as these two basins share genera and species [http://www.ldeo.columbia.edu/~ant/LAUB-FLEX/). This undercurrent movement is consistent with the weak signal of directional gene flow from Lau into North Fiji Basin , the lack of depth overlap between sites in Manus Basin and sites in North Fiji and Lau Basin, or by a yet to be determined oceanographic feature.While  centres . In this species . Water min Table . Likewiso basins . The barIfremeria nautilei at the patch, mound, or site levels within the Manus Basin. No differentiation was observed between samples of I. nautilei collected from North Fiji and Lau Basins, which are separated by ~1000 kilometres. The Manus Basin population of I. nautilei is isolated from that of the North Fiji and Lau Basins by an unknown process that limits contemporary gene flow.Theoretical and experimental studies suggest that spatially and temporally unstable environments favor broad dispersal capabilities ,50, whicIfremeria nautilei population structure at all but the broadest spatial scales is consistent with long-distance dispersal and the barrier to gene flow between the North Fiji and Lau Basin population and the Manus Basin population is likely extrinsic and not related to life history characteristics.Reproductive mode and larval type are often poor predictors of population structure in marine environments ,40,41. SIfremeria nautilei, high rates of gene flow among the sampled Manus Basin sites suggests that the Solwara 1 vents are likely to be repopulated from other Manus Basin localities, including South Su and Solwara 8. Monitoring of species recovery and genetic diversity as the Solwara 1 population recovers after extraction operations cease should add insight into the rates at which novel haplotypes and alleles accumulate in this species, providing a means to estimate the ages and sizes of extant populations. The Manus Basin population of I. nautilei comprises a genetically distinct unit that should be managed separately from the North Fiji/Lau population.Fine-scale spatial sampling and genetic analysis such as that used in this study can inform mitigation and best-management practices for mineral extraction at deep-sea hydrothermal vents. The Solwara 1 site is targeted for deep-sea mineral extraction . A robusIfremeria nautilei were collected from three hydrothermal vent sites in Manus Basin: Solwara 8, Solwara 1, and South Su  region was amplified with the species-specific primers COI-3 and COI-6 [2, 0.2 mM dNTP's, 0.5 μM each primer, and 1 unit Taq polymerase  in a 20 μl final volume. Reaction conditions were as follows: 94°C for 1 minute; 30 cycles of 92°C for 40 s, 50°C for 60 s, 72°C for 90 s; final extension of 72°C for 5 min. Amplicons were verified on 1.8% agarose gels. To remove unincorporated nucleotides, 14 μl of PCR product was incubated with 0.2 μl 10 × ExoAP buffer , 0.05 μl Antarctic Phosphatase , 0.05 μl Exonuclease I  at 37°C for 60 min followed by 85°C for 15 min to inactivate the enzymes. Bi-directional sequencing reactions were performed using the manufacturer's protocol for Big Dye Terminator Reaction . Sequenced PCR product was purified using AMPure magnetic bead system  following manufacturer's protocol, analyzed on an ABI 3730xl DNA Analyzer , and edited with Sequencher version 4.7 . Consensus sequences were compared against the NCBI GenBank database to confirm species identity [JQ074110 to JQ074134; Manus Basin haplotypes - accession # JQ074135 to JQ074170).The mitochondrial nd COI-6  as folloidentity  and aligidentity . A sequeAlviniconcha sp. 2 as an outgroup. Statistical-parsimony networks were assembled in TCS version 1.21 , nucleotide diversity (π), Fu's sF, and pairwise φST.Neighbor-joining phylograms of aligned mitochondrial sequences were assembled in MEGA version 4  with an ettings; ). Arlequettings;  was usedIfr040, Ifr043, Ifr052, Ifr068, Ifr078, Ifr086, Ifr093, Ifr094, and Ifr103) were amplified from Manus, North Fiji, and Lau Basin samples following methods reported in Thaler et al. [ID and PSIB) were used to indicate whether a given set of microsatellites contains sufficient information to be useful for assessing population structure [ID and PSIB were calculated for all useful sets of microsatellite markers . Permutr et al. ). Departr et al. ). Microstructure ,67. PID ST and FST at various nested scales from patch to basin. Microsatellite Analyser  returned by Structure. The program LDNe [Arlequin version 3.11 was used to conduct analysis of molecular variance (AMOVA). HIERFSTAT ,70 was uer  was useer , effective population size (θ), and divergence time (τ) between populations were estimated using the coalescent-based isolation-with-migration model implemented in IMa [COI in I. nautilei, so splitting time was calibrated against two hypothetical rates: μ1 = 5 × 10-8 , and μ2rea, see ). IMa ruanalyses .m = 100, and τ = 1.5, in a 40 chain geometric model. Effective sample size, autocorrelations, and trend plots were monitored to evaluate convergence. Three independent runs were compared to ensure that marginal posterior distributions had achieved similar solutions and results were averaged across the three runs. Generated trees were analysed in L-mode for best fit (default settings) and the most likely model was determined using Akaike Information Criterion [A series of short  IMa runs was conducted to optimize model parameters and determine the efficient priors for full runs. Prior probabilities and heating schemes were established at θ = 100, riterion  and 2LRRCLVD, JC, TFS, and ADT conceived the study; ADT, TFS and RCV collected samples; ADT, KZ, WS, JC, and TFS undertook molecular benchwork and analyses; ADT, CC, RCV, KZ, and CLVD drafted the manuscript. All authors have read and approved the final manuscript.Table S1 - Summary statistics for nine microsatellite loci amplified from populations of Ifremeria nautilei within Manus Basin. n = number of individuals, a = number of alleles, Rs = allelic richness, A P= number of private alleles, HE = expected heterozygosity HO = observed heterozygosity . Patches that contained only 1 allele not shown. Table S2 - Summary statistics for eight microsatellite loci amplified from populations of Ifremeria nautilei from Manus, North Fiji, and Lau Basin. n = number of individuals, a = number of alleles, Rs = allelic richness, A P= number of private alleles, HE = expected heterozygosity HO = observed heterozygosity . Patches that contained only 1 allele not shown.Click here for fileFigure S1 - Posterior probability densities for migration of Ifremeria nautilei between basins in the western Pacific, based on mitochondrial COI gene region.Click here for file


Passer domesticus) populations along an urban gradient. In line with theoretical expectations, FA was inversely correlated with genetic diversity estimated at genome level. However, this relationship was largely driven by variation at a single key locus. Contrary to our expectations, relationships between FA and genetic diversity were not stronger in individuals from urban populations that experience higher nutritional stress. We conclude that loss of genetic diversity adversely affects developmental stability in P. domesticus, and more generally, that the molecular basis of developmental stability may involve complex interactions between local and genome-wide effects. Further study on the relative effects of single-locus and genome-wide effects on the developmental stability of populations with different genetic properties is therefore needed.Fluctuating asymmetry (FA), a measure of developmental instability, has been hypothesized to increase with genetic stress. Despite numerous studies providing empirical evidence for associations between FA and genome-wide properties such as multi-locus heterozygosity, support for single-locus effects remains scant. Here we test if, and to what extent, FA co-varies with single- and multilocus markers of genetic diversity in house sparrow ( Developmental stability refers to the ability of an organism to achieve a phenotypic endpoint, predetermined by its genotype and the environment, along a developmental pathway in the face of random perturbations Gambusia holbrookiTurdus helleriLychnis viscariaPopulation and individual levels of developmental stability are most commonly estimated by corresponding levels of fluctuating asymmetry (FA), i.e. small, random deviations from perfect left-right symmetry in bilateral traits heterozygosity hypothesis states that individuals with high levels of protein heterozygosity are developmentally stable as a result of dominance or overdominance effects genomic co-adaptation hypothesis states that balanced co-adapted gene complexes result in higher developmental stability because natural selection favours alleles at many different loci that ‘harmoniously’ interact during the developmental process to produce stable phenotypes Based on developmental and genetic theory, at least two hypothetical mechanisms underlying the genetic basis of developmental stability have been put forward sensuOncorhynchus mykiss) showed reduced levels of developmental stability in heterozygotes, probably due to a reduction in enzyme activity despite potential beneficial effects of chromosomal heterozygosity Lucilia cuprina) showed that developmental stability in bristle numbers (but not wing characters) initially decreased upon exposure to a new pesticide but restored after modification of the genetic background through natural selection Drosophila melanogaster and Arabidopsis thalianaWhile numerous studies have provided empirical evidence for associations between developmental stability and genome-wide processes such as multi-locus heterozygosity, evidence for single locus effects  populations along an urban gradient. Despite the wealth of analytic tools developed for non-coding neutral markers and their presumed suitability to test relationships with genetic diversity, few studies have applied such markers to model single- and multi-locus relationships with developmental stability Here we study how developmental stability in a metric trait co-varies with indices of genome-wide and single-locus genetic diversity in microsatellite markers, within and among 26 house sparrow .2 on the ground) and ranged between 0–0.10 , 0.11–0.30 (‘suburban’) and larger than 0.30 (‘urban’) (Arcgis version 9.2.). We selected 26 plots along this gradient  and its suburban periphery to the rural village of Zomergem, located ca. 12 km NW of Ghent. Urbanization was measured as the ratio of built-up to total grid cell area  and resulted in strong signal-to-noise ratios . Fourth, we calculated unbiased signed FA values (subject specific slope deviations from the fixed regression represented the amount of asymmetry after correcting for DA and ME). Finally, we calculated absolute values of the signed FA values  for hypothesis testing. These individual estimates were used for individual-based analyses while population mean values were used for analyses conducted at the population level.Fifth, we compared the kurtosis levels of the signed FA values to detect antisymmetry To estimate FA in tarsus length at an individual and population level, we carried out mixed regression analysis with restricted maximum likelihood parameter estimation (REML) to obtain unbiased individual FA estimates Genomic DNA was extracted from ten plucked body feathers using a Chelex resin-based method  e2, Because the genotyping of noninvasive DNA samples is potentially prone to artefacts http://www.r-project.org), which also provides two additional heterozygosity-based indices, i.e. homozygosity by loci    and resup<0.001) , only rehttp://genetics.forestry.ubc.ca/ritland/programs.html) and calculated as i and l represent alleles and loci, respectively; ilS equals 1 if both alleles are allele i or 0 otherwise, ilP is the frequency of allele i at locus l and ln denotes the number of alleles at locus lRitland estimates were obtained from the software program MARK  and the relationship between genetic variability and strength, measured as total variance explained, of these (single-locus) genotype-FA associations was assessed. Positive Spearman rank correlation coefficients imply that more heterozygous markers are more informative We used Pearson correlation coefficients to quantify the strength of associations between d2-MLH: r = 0.52, p<0.001; d2-25,502 = 2.35, p = 0.0003 and F25,502 = 2.00, p = 0.0031) while d2 showed a near-significant trend .All loci were highly polymorphic and most locus by population combinations were in Hardy-Weinberg equilibrium, yet some deviations reached significance after Bonferroni correction . There was no evidence that scoring errors due to large allele drop-out or stutter contributed to this nonequilibrium. To ascertain these deviations did not influence our results we ran all analyses with and without these five markers. Removing these loci did not alter any of the overall conclusions, hence only results based on the total dataset are reported. There was no evidence for linkage disequilibrium between any pair of loci. Standard statistics for each marker are presented in p<0.001) . MLH was1,517 = 6.59, p = 0.01, R2 = 0.049; 1,517 = 4.70, p = 0.03, R2 = 0.046) . As opposed to the weak associations measured at the individual level, mean values of MLH and 1,24 = 12.31, p = 0.001, R2 = 0.34; 1,24 = 7.88, p = 0.009, R2 = 0.25) .Genetic diversity estimated by MLH and = 0.046) . When te = 0.25) ; Table 22≤0.06). When analyzing each microsatellite locus separately, the association between heterozygosity and FA at population level was strongest at loci Pdoµ1, Pdo16 and TG04-012, whereas mean differences in allelic size were strongest at locus Pdo16. After sequential Bonferroni correction for multiple testing, the association at locus Pdoµ1 remained significant . The strength of single-locus FA-d2 relationships (16 loci) were positively correlated with expected heterozygosity at population level  but not at individual level . In contrast, FA-MLH relationships did not significantly vary with genetic diversity   .2,513 = 4.25, p = 0.01): both variables were inversely related in rural populations , but unrelated in urban  and suburban  ones. In contrast, the strength and direction of FA- 2,41.6 = 0.79, p = 0.46) and FA-d2  relationships did not vary with urbanization.The strength of FA-MLH relationships tested at the individual level significantly varied with urbanization  genome and the position of the nearest known zebra finch gene.(PDF)Click here for additional data file.


Loxodonta cyclotis, which are often difficult to observe due to forest habitat. We tested the hypothesis that genetic similarity will decline with increasing geographic distance, as we expect kin to be in closer proximity, using spatial autocorrelation analyses and Tau Kr tests. Associations between individuals were investigated through a non-invasive genetic capture-recapture approach using network models, and were predicted to be more extensive than the small groups found in observational studies, similar to fission-fusion sociality found in African savanna (Loxodonta africana) and Asian (Elephas maximus) species. Dung samples were collected in Lopé National Park, Gabon in 2008 and 2010 and genotyped at 10 microsatellite loci, genetically sexed, and sequenced at the mitochondrial DNA control region. We conducted analyses on samples collected at three different temporal scales: a day, within six-day sampling sessions, and within each year. Spatial autocorrelation and Tau Kr tests revealed genetic structure, but results were weak and inconsistent between sampling sessions. Positive spatial autocorrelation was found in distance classes of 0–5 km, and was strongest for the single day session. Despite weak genetic structure, individuals within groups were significantly more related to each other than to individuals between groups. Social networks revealed some components to have large, extensive groups of up to 22 individuals, and most groups were composed of individuals of the same matriline. Although fine-scale population genetic structure was weak, forest elephants are typically found in groups consisting of kin and based on matrilines, with some individuals having more associates than observed from group sizes alone.Spatial patterns of relatedness within animal populations are important in the evolution of mating and social systems, and have the potential to reveal information on species that are difficult to observe in the wild. This study examines the fine-scale genetic structure and connectivity of groups within African forest elephants,  Spatial patterns of relatedness between individuals have important evolutionary consequences, as they influence the formation of mating and social systems within a species Mammals typically have male-mediated sex-biased dispersal while females are philopatric, creating the potential for matrilocal social groups Loxodonta africana) not only have fission-fusion sociality, but also differ from classic breeding groups because males are not associated with one matrilocal group, but search for mates across the population. Female groups change size over time and space, with some aggregations reaching hundreds of elephants Species with less rigid or dynamic social systems can still have genetic structure, and even non-social species may exhibit isolation by distance. However, patterns in such species may not be as pronounced as those in species with polygynous breeding groups. For example, species with larger groups such as herd-living ungulates include more members and a larger proportion of the genetic diversity present in the population, weakening genetic patterns Loxodonta cyclotis), a species that is not well understood due to the difficulty of conducting studies in its remote and heavily forested habitat. Characteristics of fission-fusion sociality have been detected Here we examine the fine-scale genetic structure of the African forest elephant  makes up 3% of the park , but has higher elephant densities with approximately 3.0 elephants/km2Field research was conducted in the Station d'Etudes des Gorilles et Chimpanzees (SEGC) study zone of Lopé National Park (LNP), Gabon  under pe2 sections (n = 196) according to Universal Transverse Mercator (UTM) gridlines. Sampling sessions were chosen to capture the range of seasonality. There are two dry seasons, June-August, and a less defined one from December-February. Wet seasons occur in October/November and March/April, although variation occurs between years. Our study included four, five-to-six-day sampling sessions ; session 2: October 20th–24th, 2008 (wet season); session 3: March 21st–27th, 2010 (beginning of wet season); and session 4: May 10th–15th, 2010 . Rainfall data were collected daily at SEGC. Sampling sessions were conducted within short time frames to reduce the potential for individuals to move large distances and therefore to obtain dung sample locations that more accurately represent elephant positions in relation to one another.The SEGC study zone was divided into 1.0 kmsessions ; session2 sections were searched for fresh (≤24 hours) elephant dung. Sections were chosen randomly each day and teams searched simultaneously in different sections throughout the study zone. Once a section was sampled, the section was not re-sampled during the same session to capture the largest spatial extent. Some samples were collected opportunistically; for example if found on the road while driving to a section. In forested sections, teams searched on and around known elephant trails. In savanna sections, teams searched on elephant paths identified by freshly broken vegetation. If no recent signs were observed, the section was abandoned. Each team included at least one experienced Gabonese field assistant.Within each sampling session, between 47–61 1.0 kmElephant dung can remain visible for months, but changes in appearance and odor make it possible to discern fresh samples DNA was extracted using QIAamp Mini Stool Kits (QIAGEN) and modifications in Archie et al. We genotyped samples using 12 polymorphic microsatellite loci: FH60R, FH94R, FH48R, FH19R, LA6R, LafMS02R Samples were amplified in single locus reactions or in four multiplex reactions. Single locus reactions contained 0.5 U AmpliTaq Gold Polymerase (Applied Biosystems), 1X PCR Gold Buffer (Applied Biosystems), 0.4 µM fluorescently labeled forward primer, 0.4 µM unlabeled reverse primer, 2 mM MgCl2, 0.2 mM each dNTP, 1.5 µl 20X BSA, and 3 µl DNA extract in 25 µL reactions. Thermocycling consisted of 95°C for 10 minutes, 45 cycles denaturation at 95°C for 1 minute, primer annealing at locus specific temperatures for 1 minute, and primer extension at 72°C for 1 minute, followed by an extension of 72°C for 10 minutes. Samples amplified at either 58°C or 60°C and were arranged into four multiplex reactions . Loci LaeneMarker v1.6 (Soft Genetics LLC). Samples were amplified in PCR reactions separately and scored at each locus at least two to three times. Matching heterozygotes from the same sample were scored at least twice and matching homozygotes were scored at least three times to obtain a consensus genotype Fragment analysis was performed using an ABI 3730 DNA Analyzer with Liz 600 size standard (Applied Biosystems) and genotypes were scored in Gsib, the power to differentiate between siblings enAlEx version 6.41 sib test over PIDrandom test because it is more conservative and elephants may be found in groups of related individuals sib = 0.002) were included in the analyses. PIDsib was recalculated once all samples were genotyped, and results did not change. Locus LafMS02R did not amplify reliably and was removed.We calculated PIDeliotypeGenotyping error rate was calculated using 25 randomly selected samples in RropoutWe used Dfailure to amplify the Y-chromosome) may occur in bands in samples from males, three independent runs confirmed females, while males were confirmed twice.Sex was determined following methods in Munshi-South et al. equencher 4.5 (Gene Codes Corporation). Haplotypes were identified by at least one base pair difference.We amplified a 627 bp fragment of the mtDNA control region for all individuals identified through unique genotypes using the primers MDL3 and MDL5 We used the average circumferences of three dung boli to establish age classes of juvenile (pre-reproductive) and adult (reproductive) individuals. Eggert et al. icro-Checker version 2.2.3 enepop version 4.0 We used MenAlEx version 6.41 To test if genetic similarity declines with increasing geographic distance, we conducted spatial autocorrelation (SA) analyses for adult females using Gwww.spatialecology.com/htools/) in ArcGIS 9.2  with more than two locations. Each SA was run with 9,999 permutations and bootstraps. Genetic distances were calculated from genotypes and converted to the autocorrelation coefficient r, which although not the same as, yields similar estimates to Queller & Goodnight's RSpatial autocorrelation analyses are based on a single location per individual. For repeat captures of individuals within a sampling session, we calculated a midpoint between two locations, or created centroids using minimum convex polygons with Hawth's tools v3.2  the 95% error about r generated from bootstrap tests does not intercept the x-axis at r = 0. The latter is more conservative and will favor the null hypothesis more frequently r will decrease as distance size classes increase. The first distance class where r is not significant designates the spatial extent of genetic structure in the population r was no longer significant Significance in SA can be detected if (1) oancestry version 1.0 r = 0.911). We calculated pairwise relatedness (R) in Relatedness version 5.0.8 No relatedness estimator outperforms others, and an estimator is data dependent r test atrixtester (www.rug.nl/fmns-research/beso/_people/hemelrijk) with 10,000 permutations. Matrixtester allows for matrices containing dyads of ≤100 individuals, and therefore the year analyses for 2008 and 2010 were excluded because recaptures increased matrix size.To assess the consequences of using centroids for recaptured locations from the same individual, we conducted permutation tests between matrices of dyads that compared spatial proximity with relatedness, but allowed for nonindependent data points. We used Hemelrijk's Tau KrcGIS 9.2. We chose 0.2 to ensure the following relationships would be captured; mother-daughter (R = 0.5), full siblings (R = 0.5), half siblings (R = 0.25), and grandmother-granddaughter (R = 0.25), as the mean expected relatedness value on average is 0.25, but can be lower or higher. Mitochondrial DNA haplotypes were mapped into ArcGIS 9.2 to examine the distribution of matrilines.To understand how kin are positioned in space, we calculated average distances between related adult females in week and single day sessions. Dyads with a relatedness value of at least 0.2 had their Euclidean distances measured and averaged in ATo investigate associations between individuals, we examined relatedness within and between groups of dung samples, and created network models from samples collected in groups. Samples were considered to be from the same group if they were collected on the same day, within 250 m of each other for each pair of samples, and were of the same freshness. For most samples, identifying groups was apparent, as it was rare to find numerous dung piles that were less than 24 hours old in the same area and of varying freshness. To be conservative, if there was any doubt that dung samples belonged to the same group, they were assigned to different groups.erm version 1.0 with 10,000 permutations and 10 iterations oancestry using 10,000 bootstraps.We tested if individuals within a group were more related compared to individuals from other groups using permutation tests in Pcinet version 6.403 etDraw version 2.120 elatedness version 5.0.8.We created network models from the genetic information from dung samples collected together, excluding samples found solitarily, and therefore not associated with any group. Networks were created in UWe collected 501 dung samples and identified 89 unique adult females, 22 juvenile females, 16 females of unknown age, 18 adult males, 22 juvenile males, and 10 males of unknown age from genotypes. On average, individuals were recaptured 2.249 times with a range of 0–19 recaptures. Between 2008 and 2010, 23 adult females were re-sampled.After applying a standard Bonferonni correction for multiple tests, all loci except LaT05 conformed to Hardy-Weinberg equilibrium expectations. This locus showed evidence of large allelic dropout and null alleles, and because it did not amplify consistently, removing it from analyses did not affect the number of genotyped individuals. The average number of alleles per locus was 12±3.9 and the mean observed heterozygosity was 0.820±0.081 . The aveMitochondrial haplotype diversity was 0.805±0.017 and nucleotide diversity averaged over all loci was 0.008±0.004. Ten haplotypes were identified, all of which differed from those previously reported of the same length (GenBank accession #KF938592-938601). One haplotype (Lope10) was unique to males.r tests revealed slightly different results with the significance of non-independent data between spatial proximity and relatedness occurring in session 1 , 4 , the single day , approaching significance in session 2 , but none in session 3 .Sessions 2, 4, the single day analysis, and both years revealed significant positive genetic structure at 0–5 km and negative structure at 5–10 km for adult females , 3. SpatThe mean distance between adult females with a relatedness value of ≥0.2 was 2.984±2.105 km (session 1), 2.208±1.709 km (session 2), 2.599±0.747 km (session 3), and 1.688±1.775 (session 4). The single day had a smaller mean distance of 0.823±0.548 km. The north-south sampling extent of the single day analysis was slightly smaller than the regular sampling sessions (9 km compared to a maximum of 13–14 km); however the east-west spatial extent was the same (7 km compared to 7–8 km). In all sessions, we found dyads that were closer to each other in distance, but less closely related.We detected ten mtDNA haplotypes. Lope7 was the most common, but individuals with different haplotypes were found in close proximity (<1.0 km), and overall no spatial segregation was observed. The single day session yielded the same pattern, with samples collected <100 m from each other having different haplotypes.R = 0.236±0.187, p<0.001) and all individuals . In 84.6% of groups (22 of 26), adult females had the same mitochondrial haplotype, and in 82.9% of groups (58 of 70), all individuals shared the same mitochondrial haplotype .We detected 70 groups and 26 included more than one adult female . Individaplotype . ConsistThe network included 106 individuals with 28 components and 83 edges . The larr test, which tests for the relationship between relatedness and distance, but accounts for non-independent repeat locations. Spatial autocorrelation analyses revealed that adult females are more closely related than expected by chance from 0–5 km in two of the four week sampling sessions, samples collected over four-months in 2008 and 2010, and in the single-day analysis. No genetic structure was found in session 1, but there was a significant correlation between distance and relatedness in the Tau Kr test. Alternatively, significant genetic structure was detected in sampling session 2, but not in the Tau Kr test, although it approached significance (p = 0.055). Conflicting results between the two methods may be due to this test's use of recaptures and not midpoints.This study revealed fine-scale genetic structure, albeit weak, and evidence of potentially complex, kin-based groups using a novel network model genetic approach derived from non-invasive dung sampling in African forest elephants. We tested the hypothesis that genetic similarity decreases with increasing geographic distance for two types of spatial genetic patterns; spatial autocorrelation (SA) analyses in which distance was separated into classes of 5 km, and the Tau KDiceros bicornis bicornisMeles melesRattus fuscipesSignificant SA coefficients were 0.014 or lower, and for inclusive distance classes, 0.083 or less within the first distance class. Although significance was detected, compared to other mammals , individuals may not be placed in the distance class that more accurately represents their true positions relative to one another at that time. The only session to yield significant positive genetic structure with all analyses, and in all of the inclusive distance classes up to five kilometers, was the single day session. This suggests that the time involved in sampling is likely an important factor in capturing fine-scale spatial genetic patterns of forest elephants. The single day analysis includes recaptures, but likely reduced the effects of movements because of the shorter time scale. However, reducing sampling time also limits sample size. To examine these effects, we increased sample size by using data from dung samples collected for each year. Both 2008 and 2010 yielded similar patterns with significant positive genetic structure in the 0–5 km distance class and negative in the 5–10 km class. Future studies should therefore concentrate on increasing sampling effort over shorter time periods.When looking at the spatial patterns of related dyads , related adult females were found within several kilometers of each other. However, it was not uncommon to find more geographically distant dyads more closely related than individuals that were closer together, even for samples collected on the same day. There was no spatial structure associated with mitochondrial haplotypes, and based on dung locations and dates, individuals appeared to tolerate others with different haplotypes in close proximity. This may explain the weak genetic structure from the SA analyses and inconsistent results between sampling sessions. Although we detected a signal of positive genetic structure within five kilometers, it is likely diluted by the presence of unrelated individuals in the area. However, consistent with evidence of male-biased dispersal that typically occurs in mammals, females had a significantly higher mean pairwise relatedness than males in the population.Sacoglottis gabonensis, with dung rates highest in September – November. Our study found genetic structure through SA in only one of the two of the sampling sessions that occurred in the months with both the most and fewest number of dung piles. Therefore, genetic structure can be detected, even when densities are high, however, higher densities also have the potential to dilute patterns, necessitating the importance of looking at individual association patterns.The SEGC study zone differs from most forest elephant habitat because it is a savanna-forest mosaic and also has slightly higher elephant densities r test. In contrast, session 3 had the second highest amount of rainfall, yet no spatial genetic structure was detected.We also did not find any relationship between rainfall and the results of spatial genetic analyses. Session 2 occurred during the month with the most rainfall, and we found significant SA, and an almost significant negative relationship between relatedness and spatial proximity in the Tau KOther factors possibly contributing to inconsistent results may include social structure, poaching, and sample size. Evidence is mounting that forest elephants have fission-fusion sociality, where group sizes and composition change over time Despite weak genetic structure, we found relatedness in dung samples collected as groups to be consistent with family group expectations. Individuals within groups were significantly more related to each other than to individuals from other groups, and average pairwise relatedness between adult female elephants (0.236) was comparable to family groups in savanna elephants Click here for additional data file.Table S2R) and 95% confidence intervals (CI) within groups.Group composition, mitochondrial haplotypes, average pairwise relatedness Click here for additional data file.


Mysis salemaai collected at night in the pelagia and close to the bottom. Stable isotope signatures were significantly different between migrants (pelagic samples) and residents (benthic samples), indicating persistent diet differences, with pelagic mysids having a more uniform and carnivorous diet. Sequencing of the mitochondrial cytochrome subunit I (COI) gene showed genetic differentiation attributable to geographic location but not between benthic and pelagic groups. Divergent migration strategies were however supported by significantly lower gene flow between benthic populations indicating that these groups have a lower predisposition for horizontal migrations compared to pelagic ones. Different migration strategies did not convey measurable growth benefits as pelagic and benthic mysids had similar growth condition indices. Thus, the combination of ecological, biochemical and genetic markers indicate that this partial migration may be a plastic behavioral trait that yields equal growth benefits.Diel vertical migration (DVM) is often assumed to encompass an entire population. However, bimodal nighttime vertical distributions have been observed in various taxa. Mysid shrimp populations also display this pattern with one group concentrated in the pelagia and the other near the bottom. This may indicate alternative migratory strategies, resembling the seasonal partial migrations seen in birds, fishes and amphibians, where only a subset of the population migrates. To assess the persistence of these alternative strategies, we analyzed the nitrogen and carbon stable isotope signatures (as proxies for diet), biochemical indices (as proxies for growth condition), and genetic population divergence in the Baltic mysid  In aquatic ecosystems, processes that mediate energy flux between benthic and pelagic environments are crucial in maintaining ecosystem integrity and seasonal productivity Mysid shrimps  are common in both marine and freshwater systems. They have strongly pronounced DVM and are capable of migrating several hundreds of meters from the bottom towards the surface at night Mysis mixta Liljeborg, Mysis relicta Lovén and Mysis salemaai Audzijonyte and Väinölä M. relicta and M. salemaai are sympatric M. salemaai was until recently unknown and grouped with M. relicta, as were two other cryptic species from North America . This implies that earlier reports on M. relicta may in fact refer to a mixture of sibling species, in all areas where the cryptic species co-occur.In the Baltic Sea, there are three species of pelagic mysids: Even though DVM is a prominent feature of mysid behavior, a bimodal night-time vertical distribution in mysid populations has been observed  and thus decrease intraspecific competition Although partial migrations, where animals adopt different life history strategies (migration or residency) have been described in seasonal environments, few studies have considered the possibility of such a migratory strategy in cases where migration is considered to be more or less regular, like DVM. Recently, Mehner and Kasprzak Mysis spp. in concert with their diet and growth condition in relation to their migratory behavior (pelagic vs. benthic) in a coastal area of the Baltic Sea. Once the absolute dominance of M. salemaai in both pelagic and benthic groups was established, we used mitochondrial DNA to assess the genetic structure of our sampled population. To assay feeding habitats of the mysids, we used a stable isotope approach, a useful tool in studying food web interactions and movements between isotopically distinct food webs 15N) compared to their diet and this can be used to estimate trophic position within a food web 15N and δ13C. To assess differences in mysid growth condition between the habitats and stations, we used the protein:DNA ratio in individual mysids; this ratio has been successfully used as a surrogate marker of changes in individual growth related to cell size dynamics and gross protein synthetic activity in different crustaceans, including mysids (M. diluviana) Here, we examine whether the observed bimodal night-time distribution in mysids can be explained by (i) interspecific differences in migratory behavior between the sibling species, (ii) an unsynchronized DVM within the homogenous population, i.e. non simultaneous migration towards the surface, or (iii) consistent, intraspecific divergent migratory tactics with distinct genetic morphs, thus resembling the partial migrations undertaken by many species of fish and birds. We address these hypotheses by examining the species composition of 15N, resulting in elevated concentrations of this isotope in producers and consumers of the food web, whereas Station S1 is not measurably affected by discharges from the sewage treatment plant Mysids were sampled at two coastal stations  at a depth of 25±2 m. Benthic mysids, occurring 5–30 cm above the sediment surface, were collected with an epibenthic sled Mysis relicta/salemaai were separated from other mysids based on the morphological characteristics described by Mauchline Samples were collected at night (22:00 to 04:00), 8-9-Sept-2008. No daytime samples were taken, because Mysis relicta and M. salemaai were identified by amplification and sequencing of a 634 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. ®) was added to homogenized pleopod samples and heated for two hours at 60°C followed by 105°C for 10 min Mysis primers: myshatD1516, 5′-cactctattttgtttttggrgcttg- 3′and myshatR2164, 5′-cttctgccaccttctttagctc- 3′ ® DNA Analyzer  using both the forward and the reverse primers (24 samples), which resulted in identical sequences, or only the reverse primer (rest of the samples). To assess sequencing error, 15 individuals were run twice and were invariably assigned to the same haplotype.http://www.ddbj.nig.ac.jp/search/blast-j.html) using the NCBI BLAST function optimized for highly similar sequences (megablast) for species identification based on the highest alignment score.The sequences were visually inspected, aligned and trimmed in BioEdit v. 7. 0. 5. 3, resulting in a 469 bp sequence of sufficient quality used for further analysis. These sequences were subjected to a BLAST search  using a caliper (±0.02 mm).All mysids were sexed according to the sexual characteristics described in Mauchline MysisFor SIA, both muscle tissue and purified chitin were used. Muscle tissue has a relatively low turnover and reflects the diet over most of the growth season in subadult Samples were analyzed for their relative abundance of stable carbon and nitrogen isotopes as well as per cent carbon and nitrogen using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer . Isotopic analyses were performed at UC Davis Stable Isotope Facility, University of California, Davis. The results are expressed in δ as parts per thousand (‰) according to equation 115N or 13C and R is the corresponding ratio 15N/14N or 13C/12C. The reference material used was atmospheric N2 and PeeDee Belemnite, respectively. Two working standards (homogenized mysid tissue) were run for every 42±2 samples (n = 28). The analytical precision for δ 15N and δ 13C were within the limits of±0.13‰ and±0.09‰ S. D. respectively. Analytical and method blanks were also included to control for possible analytical drift as well as contamination risk during laboratory work. No lipid correction was necessary as C:N ratio never exceeded 3.5  were determined using microplate-based bicinchoninic acid assay  with bovine serum albumin as standard and fluorometric high-range RiboGreen  assay with calf thymus DNA as standard Pleopods were dissected from ethanol-preserved mysids (8 to 18 per station/habitat group) and used to quantify total water soluble protein and DNA. The samples were homogenized in 130 µL of 1% N-laurylsarcosine using a FastPrep homogenizer with cooling function, and incubated for 2 h on a shaker. The protein and DNA concentrations  to test for effects of body size and differences between the benthic and pelagic samples on a combination of carbon and nitrogen isotopic ratios. We then used a factorial analysis of variance/covariance  of the ‘nlme’ package in R STFWe assessed global and pairwise genetic differentiation using Fisher's exact tests with 10 000 dememorisation steps and an analysis of molecular variance (AMOVA) based on genetic distances between haplotypes STF values observed, the statistical power of our analysis to reject the null hypothesis of genetic homogeneity was assessed by a power-test (POWSIM) STF = 0.01. We used the default settings in POWSIM, modeling over 10 generations (t) and with an effective population size (eN) of 500. The analysis showed that our dataset had high power (97.6%) of detecting global population structure even at a low level of genetic differentiation (STF = 0.01).Given the relatively low M. salemaai , with a few M. relicta (n = 8) that were all caught pelagically. Therefore, the analyses of stable isotope signatures and population genetic structure reported here are focused exclusively on M. salemaai.The mysid community was heavily dominated by  = 81, p = 0.017).Mysids analyzed for stable isotopes all consisted of juveniles and immature males where the latter contributed 20–48% of the total sample size and were similar in size with a mean BL of 14.06±0.78 mm (±S.D.). However, individuals in the benthic group from station S2 were on average 0.8 mm smaller than individuals in the pelagic group , which explains the significant interaction between station and BL  and 0.6‰ (δ13C) higher at station S2 , with adjusted mean δ13C being 0.15‰ higher in the pelagic zone. A tendency, albeit not significant, towards lower (muscle tissue) and higher (chitin) δ13C values in benthic compared to pelagic samples were observed , whereas benthic correlations were considerably weaker .In the pelagic habitats, correlations between δt1, 52, = 2.6, p = 0.012) but not between the habitats  and these results were not affected by BL . The adjusted mean of log protein  was slightly higher at station S2 indicating a higher growth status .There was a significant difference in protein:DNA ratio between the stations  with slightly lower ratios at station S1, whereas no difference was found between the habitats . C:N ratios were not affected by mysid size .C:N ratios differed significantly between the stations , six of which  have been described previously A total of 33 mtDNA haplotypes were identified among the 160 individuals of Minimum spanning tree showing the relationship between haplotypes found in pelagic and benthic samples at station S1 and S2. The size of the circles is proportional to the relative frequency of haplotypes. The smallest colored circle represents one individual. Grey circles show missing or not sampled haplotypes.p>0.09), the panmixia hypothesis was rejected based on low yet significant global genetic differentiation  as well as between the pelagic group at station S2 and the benthic group at station S1 (STF = 0.054). All other pairwise comparisons were non-significant ]. The summer average δ15N-signal of seston  was approximately 3 and 4.2‰ at sites that are in close proximity to stations S1 and S2 respectively, demonstrating that the δ15N discrepancy observed between mysids at stations S1 and S2 was due to station specific differences in baseline and not a result of different diets.As expected, due to enrichment in 15N values between the benthic and pelagic groups suggest consistent time-integrated differences in diet, both on the longer and shorter time scale. Muscle tissue signatures have a turnover of 2–3 months in mysids of this size 13C at station S1 did not differ significantly between the habitats may indicate more a lack of difference in carbon isotopic composition of the ingested food between the habitats rather than similar diets. In omnivores, a true difference in diet can be hidden, because their isotopic signature often reflects that of their dietary protein. Thus, even a low intake of animal tissue in benthic mysids would produce relatively high imprints on the δ-values, suggesting a diet more similar to that of pelagic mysids than in reality. For example, omnivorous crayfish display trophic positions close to those of predatory fish –which is higher than would be expected from gut content analysis alone 13C between the groups at station S1. The more carnivorous nature of pelagic mysids 15N, δ13C-correlations as compared to benthic individuals that appear to have broader, more omnivorous diets. Polunin et al.et al.Between-habitat differences were consistent, with both pelagic groups having higher average values than respective benthic groups, both in muscle tissue and chitin. The small, yet significant, differences in δM. salemaai populations, particularly between the bottom-collected animals. When accounting for the amount of molecular differentiation between the haplotypes (AMOVA), a significant fine-scale genetic structure was observed, with most haplotypes being unique to their respective sample locations. The most important observation is that in the continuous population of M. salemaai, bottom-dwelling mysids have a significant degree of genetic differentiation between the closely situated sites (∼20 km). Moreover, benthic mysids at station S1 had the lowest within-group diversity and were significantly different from not only the other benthic, but also the pelagic group at station S2. The differences observed are most likely related to the restricted migrations of mysids collected near the bottom at night compared to the pelagic individuals. If this is true, then pelagic individuals would contribute most to the gene flow in the population. Intuitively, this makes sense, since individuals performing extensive DVMs would be more susceptible to passive transportation by currents than non-migrating individuals.The genetic structure analysis suggests that significant genetic differentiation occurs between local Although many causes for the observed genetic differentiation within the mysid population inhabiting the area may exist, we suggest migratory behavior being an important factor affecting gene flow rate. In our study area, estuarine circulation is the main hydrodynamic mechanism accounting for the water exchange between Himmerfjärden Bay north of station S2 and the open coastal area where S1 is located M. salemaai population shows ecological niche partitioning, with two reasonably distinct ecotypes. The pelagic ecotype conducts a DVM, has higher trophic position, more uniform diet, and contributes largely to horizontal migrations and gene flow within a population, whereas the benthic ecotype is relatively sedentary, with limited (if any) DVM, is more omnivorous, presumably feeding to a larger extent on detritus, and has a spatially restricted gene flow. Undoubtedly, the patterns of mysid migration seen here strongly resemble those reported for partially migrating fishes, birds and amphibians. Although there are numerous studies demonstrating bimodal vertical zooplankton distributions The combination of ecological and genetic markers, indicate that the As we found no evidence for significant genetic differentiation between benthic and pelagic mysids within stations, it is plausible that this trait is plastic as reported for several fish species Gobidae) that primarily feed on less motile prey, such as benthic amphipods, harpacticoids and oligochaetes (unpublished data). Thus, the predation pressure from these fish is low compared to that of the much more abundant herring, which feed to a considerable extent on mysids Moreover, as mysids are proposed to be food limited even when the zooplankton abundance is at its annual maximum If the partial DVM that we have observed is evolutionary stable and yields equal fitness benefits to both strategies M. mixta having a varying size distribution pattern during the growth season: a unimodal in late spring-early summer, a bi-modal in summer-early autumn, and a unimodal again in late autumn. This dynamics in individual body size indicates diverging growth patterns, with a part of the population growing considerably faster under a period of time. Although we did not observe differences in the growth proxies in the autumn, they may occur during other periods and under different environmental conditions. Assuming that such patterns are frequent, it is possible that growth rate could be the decisive factor regulating migration in mysids as found for partially migrating fish.What determines an individual's decision to migrate or not is still debatable but seems to be linked to conditional cues, at least in fish We hypothesized that the apparent lack of migration in some mysids could have been attributed to either (i) interspecific differences in migration between the sibling species, (ii) an unsynchronized DVM within the homogenous population, i.e. non simultaneous migration towards the surface, or (iii) consistent, intraspecific divergent migratory tactics with distinct genetic morphs.M. relicta individuals were present in our samples. Persistent differences in the resource use and indications for restricted horizontal migration in the benthic part of the population were however found within the sampled M. salemaai population; demonstrating that these differences could not have arisen in a population conducting unsynchronized migrations (second hypothesis). Feeding on equal amounts of the same sources but at different times should have resulted in isotopic values more similar between benthic and pelagic mysids than those observed in our study. Finally, the lack of genetic differentiation at the level of mtDNA between the benthic and pelagic mysids suggests that the observed differences in migratory strategies are plastic. However, application of other genetic markers is needed to further investigate the genetic component of the divergent strategies and its interaction with the environment. In any case, our results provide evidence for the existence of two M. salemaai ecotypes that differ in their DVM behavior and feeding habits, without any clear differences in growth condition; thus resembling many cases of partial migration commonly observed in birds and fish populations.The first hypothesis could not be rigorously tested since too few Ignoring the existence of these ecotypes is likely to complicate e.g. food web mapping and modeling by means of stable isotopes as well as other environmental studies, such as monitoring of environmental contaminants


Sporisorium scitamineum isolates were investigated by inter simple sequence repeat (ISSR) and single primer-sequence related amplified polymorphism (SP-SRAP) markers. These isolates were clearly assorted into three distinct clusters regardless of method used: either cluster analysis or by principal component analysis (PCA) of the ISSR, SP-SRAP, or ISSR + SP-SRAP data set. The total gene diversity (Ht) and gene diversity between subpopulations (Hs) were estimated to be 0.34 to 0.38 and 0.22 to 0.29, respectively, by analyzing separately the ISSR and SP-SRAP data sets, and to be 0.26–0.36 by analyzing ISSR + SP-SRAP data set. The gene diversity attributable to differentiation among populations (Gst) was estimated to be 0.35 and 0.22, and the gene flow (Nm) was 0.94 and 1.78, respectively, when analyzing separately ISSR and SP-SRAP data set, and was 0.27 and 1.33, respectively, when analyzing ISSR + SP-SRAP data set. Our study showed that there is considerable genetic variation in the analyzed 100 isolates, and the environmental heterogeneity has played an important role for this observed high degree of variation. The genetic differentiation of sugarcane smut fungus depends to a large extent on the heterogeneity of their habitats and is the result of long-term adaptations of pathogens to their ecological environments.A total of 100  Saccharum complex) is the most important sugar-yielding crop in China and accounts for about 92% of sugar output [ Sporisorium scitamineum is one of the most intractable and devastating diseases of sugarcane in the world [China is the second largest consumer of sugar and the third largest producer internationally . Sugarcar output . It has r output . Sugarcahe world . It caushe world . At pres S. scitamineum. The investigation of the genetic diversity of S. scitamineum is essential for a better understanding of the structure of pathogen populations and the interactions among host and pathogen and the environment and as a basis for developing smut-resistant sugacane varieties. Molecular detection of the smut pathogen in sugarcane was firstly realized by using polymerase chain reaction (PCR) and then TaqMan Real-time PCR to amplify the bE mating-type gene of S. scitamineum [ S. scitamineum isolates has been studied by the methods of random amplification of polymorphic DNA (RAPD) [ S. scitamineum populations from 15 sugarcane producing countries for polymorphisms at 17 microsatellite loci [ S. scitamineum populations all belonged to a single lineage which was also found among some Asian populations. In China, Que et al. studied the molecular evolution of S. scitamineum by analyzing a set of 23 isolates collected from six primary sugarcane producing areas in Mainland China with RAPD and sequence-related amplified polymorphism (SRAP) markers [ S. scitamineum was associated with its geographical origin. No evidence of coevolution between the pathogen and sugarcane was found. These analyses also did not provide information about the race differentiation of S. scitamineum. Furthermore, the numbers of host genotypes, smut isolates, and geographical origin of districts in China for testing were limited to 21, 23, and 6, respectively, in the study of Que et al. [The smut-resistant mechanism of sugarcane has been widely investigated at the morphological, physiological, and mainly molecular level –3. Severtamineum , 18, 19.A (RAPD) , 20, ampA (RAPD) , and intA (RAPD) , 22. Rab S. scitamineum could help to identify potential sources of resistance, which are expected in areas where genetic diversity of both pathogen and host is maximal [ S. scitamineum populations was shown to be extremely low among 142 single teliospore isolates collected from 15 countries based on the analysis of AFLP and RAPD markers [ Brassica crops by Li and Quiros [Population genetic analysis of maximal . The gen markers . ISSR ma markers . Therefo markers –26. Sequd Quiros . Currentd Quiros . In our  S. scitamineum isolate collection from Mainland China with the aim of understanding, at the molecular level, the differentiation, variation, and population structure of S. scitamineum in Mainland China and the effectiveness of the environmental and genotype heterogeneity on the influence of the pathogen population dynamics to provide the basis for S. scitamineum resistance breeding and the geographical distribution of varieties.In this study, we use ISSR and SP-SRAP techniques to study a more representativeOne hundred pathogen strains were isolated in 2010 from 38 different sugarcane genotypes in 7 provinces (20 sugarcane planting districts) of China, including Guangxi (which accounts for 62% sugarcane planting area) (36 strains), Yunnan (26 strains) (accounts for about 20% sugarcane planting area), Guangdong (13 strains), Fujian (9 strains), Jiangxi (8 strains), Hainan (5 strains), and Sichuan (3 strains). More detailed information about these strains are given in μL) under the microscope (40 × 10). 10–30 μL of dilution was then evenly plated onto potato dextrose agar (PDA) medium, containing 75 mgl−1 streptomycin sulphate according to Yang et al. [Single teliospores were isolated from whips of infected sugarcane with sterile tips or inoculation needle under the sterile conditions and separated by serial 10-fold dilution until only 1–5 single-teliospore(s) were observed in a single drop  regions  as described by Singh et al. [ S. scitamineum, we amplified these DNA samples by S. scitamineum specific primers bE4 and bE8 [http://www.ncbi.nlm.nih.gov/).Genomic DNA from hyphal colonies developed from single teliospores were extracted by the modified SDS method described by Que et al. then storh et al. . To ensu and bE8 , cloned  S. scitamineum samples, we first performed PCR amplifications using the universal primers ITS1 and ITS4 for detection of fungi [bE4 and bE8 for detection of S. scitamineum [To ensure the identity of ourof fungi  and the tamineum . Ex Taq polymerase purchased from the TaKaRa Biotechnology Co., Ltd. . Amplification reactions were performed in a total volume of 20 μL containing 20 ng of DNA, 5 pmol of each primer, 4.5 nmol of each dNTP, 2.2 μL of 10 × Ex Taq Buffer, and 0.5 U Ex Taq polymerase. The final volume was adjusted to 20 μL with sterile distilled water [For ISSR analysis, the primers used  were commercially synthesis anded water , 24. TheμL containing 2.4 μL of 10 × Ex Tap Buffer, 5 nmol of each dNTP, 20 pmol of primer, 1.0 U Ex Taq polymerase, and 40 ng of DNA template. The amplification cycles consisted of an initial denaturation at 95°C for 5 min, followed by 5 cycles of 1 min at 94°C, 1 min at 35°C, and 1 min at 72°C, 35 cycles of 1 min at 94°C, 1 min at 35°C, and 1 min at 72°C, and a final extension phase of 10 min at 72°C. PCR products were separated by electrophoresis in 3.0% agarose gel with 1 × TAE buffer at 60 V for 3 h. The gels were stained with ethidium bromide and then photographed under ultraviolet light as above.For SP-SRAP analysis, the amplification reactions were performed in a volume of 25 Na), effective number of alleles (Ne), Nei's measure of gene diversity (h), and Shannon's Information index (I) were used to evaluate the genetic diversity within each population by POPGENE1.31 [Ht), Shannon's diversity index between populations (Hs), proportion of gene diversity attributable to differentiation among populations (Gst), and estimates of gene flow parameter among populations Nm = 0.5(1 − Gst)/Gst  were obtained across loci [The softwares of NTSYS.PC   and POPGGENE1.31 . Mean vaoss loci .http://www.lynnon.com/) and the consensus sequences were compared by BLASTn to the NCBI databases (http://www.ncbi.nlm.nih.gov).Based on results of ISSR and SP-SRAP analyses, somepairwise comparisons among isolates with low similarity values were selected for further investigation of ITS sequences . The sequences were analysed by using the software DNAMAN  cluster analysis and is presented in Based on the ISSR data set, a dendrogram was generated for the 100A principal component analysis (PCA) based on the ISSR data set was performed. The first three components explained 97% of the total variance and divided clearly the 100 isolates into three groups, A, B, and C, as shown in  S. scitamineum isolates and yielded a total of 153 bands, of which 152 (99.3%) were identified as polymorphic. The number of polymorphic bands generated by each primer ranged from 9 to 27 with an average of 21.7. Except the primer P2, which produced one monomorphic band (of 10 bands) among the 100 isolates, the other 6 primers produced 100% polymorphic bands  and YT94-128 (number 19), ROC22 (number 18) and YT94-128 (number 19), and YT94-128 (number 19) and ROC22 (number 20). All 4 isolates were collected from Guangxi.The genetic similarity indexes generated from SP-SRAP data for the 100 S. scitamineum isolates into three clusters, I, II, and III, at the distance index of 0.01  were polymorphic on the 100 and III . Of the  S. scitamineum isolates based on the combined ISSR + SP-SRAP data set were shown in The results of PCA analysis of the 100Compared to the results of UPGMA analysis, all 45 isolates in group A and all 23 isolates in group B were also grouped in the cluster I of UPGMA, while group C contained all four isolates in cluster II and all 20 isolates in cluster III, plus the remaining eight isolates in cluster I. The isolates isolated from the same district in a province tend to group together as revealed by both UPGMA and PCA analyses. S. scitamineum isolates were estimated (Na) and effective (Ne) numbers of alleles were higher in the population of Guangxi  and Yunnan  compared to other subpopulations. By comparison, the gene diversity index (h) and the Shannon's information index (I) were also relatively higher in the subpopulation of Yunnan  than those in the subpopulations of Hainan  and Sichuan , respectively. By using ISSR data set, the total gene diversity (Ht) and gene diversities between subpopulations (Hs) were estimated to be 0.34 and 0.22, respectively. Gene diversity and differentiation among populations (Gst) were estimated to be 0.35, indicating that 35% of the total genetic variation was originated between populations, while gene flow (Nm) was estimated to be 0.94, indicating that lower rate of gene flow has occurred between populations.Population genetic parameters for the 121 ISSR loci identified among the 100stimated . The resNa) and effective (Ne) numbers of alleles were higher in the subpopulations of Guangxi  and Yunnan  compared to other subpopulations. By comparison, the gene diversity (h) and the Shannon's information index (I) values were also relatively higher in the subpopulations of Yunnan  than those in the subpopulations of Hainan  and Sichuan , respectively. The total gene diversity (Ht) and gene diversities between subpopulations (Hs) values in SP-SRAP analysis were estimated to be 0.38 and 0.29, respectively. Gene diversity and differentiation among populations (Gst) were estimated to be 0.22, indicating that 22% of the total genetic variation was originated between populations, while the gene flow (Nm) was estimated to be 1.78, indicating that higher rate of gene flow has occurred between populations.Population genetic parameters for the 153 SP-SRAP loci identified among the 100 isolates was estimated . ObserveNa) and effective (Ne) numbers of alleles were higher in the subpopulations of Guangxi  and Yunnan  compared to other subpopulations. The gene diversity (h) and the Shannon's information index (I) values were also relatively higher in the subpopulations of Yunnan , but relatively lower in the subpopulations of Hainan  and Sichuan , respectively. The total gene diversity (Ht) and gene diversities between subpopulations (Hs) values in ISSR + SP-SRAP analysis were estimated to be 0.26 and 0.36, respectively. The genetic diversity attributable to differentiation among populations (Gst) was estimated to be 0.27, indicating that 27% of the total genetic variation was originated between populations, while the rate of gene flow (Nm) was estimated to be 1.33, relatively lower than that of SP-SRAP (1.78) but higher than that of ISSR (0.94).Population genetic parameters for the 274 ISSR + SP-SRAP loci identified among the 100 isolates was estimated . Observe S. scitamineum isolates. The PCR products from each of these 12 isolates were sequenced. Sequence analysis showed that these isolates shared 94.09% to 96.98% similarity with an average of 96.34%, suggesting that rDNA-ITS regions contain lower sequence variations and may be not suitable for studying the genetic diversity of S. scitamineum in Mainland China.A total of 12 isolates in six pairs , having low genetic similarity with the remaining pairs of the 100 isolates were selectedfor further PCR analysis of their rDNA-ITS, including ITS1, ITS2, and 5.8S rDNA, using the primer pair ITS1 and ITS4 . PCR amp S. scitamineum isolates, PCR amplifications of the ITS region were performed using the specific primer pair ITS1 and ITS4. As expected a single 755 bp fragment was obtained on each isolates. PCR amplifications with S. scitamineum—specific primer pair bE4 and bE8 yielded, as expected a single 420 bp fragment from all isolates.Because of the limited genetic variability within the ITS region, ITS sequence data can normally define organisms at the genus level and resolve relationships between closely related species , 33. In These results showed that a single spore isolated from one smut whip can be used to generate a genetically fixed clone representing its original local population. Furthermore, sequence analysis of ITS regions of the six pairs of 12 isolates with lower genetic similarity indexes (from 0.4628 to 0.5627) showed very high sequence conservation between these isolates . This result confirms a previous observation by Singh et al. [ S. scitamineum are known to exist but the race type is still poorly understood [ S. scitamineum isolates into clusters based on smut race [ S. scitamineum from different geographic areas, by the RAPD, SRAP, and AFLP methods has so far yielded data of little value to the race classification of sugarcane smut.Several races ofderstood , 34–37. derstood . Using R S. scitamineum requires further exploration.Similar to the previous study , the res S. reilianum collected from six provinces of China [ S. reilianum was associated essentially with the geographic origins of the pathogen instead of their host genotypes [ S. commune [ S. scitamineum was studied by RAPD, SRAP, SSR, and AFLP methods [ S. scitamineum and its sugarcane host, while coevolutionary phenomena between pathogens and their hosts have been proposed in Magnicellulatae and Podosphaera [ S. scitamineum tends to be associated with their geographical origins, not to their host origins. The genetic diversity within a collection of 23 isolates of S. scitamineum from Mainland China was relatively high with a genetic dissimilarity index value ranging from 0.071 to 0.825 for RAPD analysis, from 0.045 to 0.947 for SRAP analysis, and from 0.026 to 0.838 for combined RAPD-SRAP analysis [ S. scitamineum populations were extremely low in genetic diversity and all strains seem to belong to a single lineage according to a study of 142 single-teliospores using SSR markers [ S. scitamineum was 0.66 by ISSR and 0.62 by SP-SRAP. Our study showed that the genetic diversity among the Chinese smut pathogen population was relatively high and properly estimated by the two adopted methods with the SP-SRAP markers slightly more polymorphic than ISSR markers.Ma et al. studied 102 isolates ofof China . They foenotypes . The sim commune . In prev methods , 7, 9. Qosphaera , 41. In  S. scitamineum from different provinces of Mainland China and from different districts in Guangxi province which accounts for about 62% of the total sugarcane cultivated area in Mainland China. Also included in this study were 26 strains isolated from Yunnan province which is considered to be the plant genetic diversity center [ S. scitamineum.One of the aims of the present study was to estimate the genetic relationship between the subpopulations ofy center  because PCA analysis showed that the strains collected from the same region tend to be grouped together in the resulting plots Figures , and 7.  S. scitamineum isolates, that is, the isolates of the smut pathogen collected from a same region tend to be clustered in the same group. Isolates from a same province were shown to share a high genetic similarity, while the isolates from different provinces were shown to be more genetically distant with lower genetic similarity values among them  within the population of 100 isolates of smut pathogen was estimated to be 0.34, 0.22, and 0.26, respectively; the gene diversity between subpopulations (Hs) was estimated to be 0.38, 0.29, and 0.36, respectively; the gene diversity attributable to differentiation among populations (Gst) was estimated to be 0.35, 0.22, and 0.27, respectively; the gene flow (Nm) was estimated to be 0.94, 1.78, and 1.33, respectively. In each case, the estimated values of Ht and Nm are higher for SP-SRAP than for ISSR and ISSR + SP-SRAP, and the highest value of Hs was found for ISSR + SP-SRAP data set, while the highest value of Gst was found for ISSR data set. Therefore, the combined ISSR + SP-SRAP analysis should be more appropriate for the analysis of gene diversity between subpopulations (Hs) and for the analysis of gene flow (Nm), SP-SRAP works better than ISSR and ISSR + SP-SRAP data set.Population genetic analysis, which can provide strong evidence about genetic diversity at the level of population or subpopulation, should be helpful for our understanding of the genetic structure of the studied populations , 43, 44.Nm, which means the product of the effective population number N and the rate of migration among populations, reflects the degree of genetic differentiation within populations. Methods for estimating how much gene flow occurs in natural populations can be divided into two categories; one is direct methods which involve estimating dispersal distances and reproductive success of individuals that disperse by direct observation [ S. scitamineum. According to the value of proportion of gene diversity attributable to differentiation among populations (Gst), the values of gene flow parameter Nm were estimated by indirect methods to be 0.94, 1.78, and 1.33, respectively, for ISSR, SP-SRAP, and combined ISSR + SP-SRAP analysis. All three analyses showed that there does exist significant gene flow between the seven S. scitamineum subpopulations in Mainland China. In addition to environmental factors that affect patterns of gene flow at the macrogeographic and regional scales, population structuring within ecological zones and habitats can potentially ruin efforts to drive genes into wild S. scitamineum populations [The gene flow parameter ervation ; the othervation . In the ulations , 43, 44.ulations . It can ulations .


Panicum miliaceum L.) is one of the world's oldest cultivated cereals, with several lines of recent evidence indicating that it was grown in northern China from at least 10 000 cal bp. Additionally, a cluster of archaeobotanical records of P. miliaceum dated to at least 7000 cal bp exists in eastern Europe. These two centres of early records could either represent independent domestications or cross-continental movement of this cereal that would predate that of any other crop by some 2 millennia. Here, we analysed genetic diversity among 98 landrace accessions from across Eurasia using 16 microsatellite loci, to explore phylogeographic structure in the Old World range of this historically important crop. The major genetic split in the data divided the accessions into an eastern and a western grouping with an approximate boundary in northwestern China. A substantial number of accessions belonging to the ‘western’ genetic group were also found in northeastern China. Further resolution subdivided the western and eastern genepools into 2 and 4 clusters respectively, each showing clear geographic patterning. The genetic data are consistent with both the single and multiple domestication centre hypotheses and add specific detail to what these hypotheses would entail regarding the spread of broomcorn millet. Discrepancies exist between the predictions from the genetic data and the current archaeobotanical record, highlighting priorities for investigation into early farming in Central Asia.Broomcorn millet ( Phylogeographic studies of crop plants play an important role in understanding the population history of both the domesticated or cultivated plants themselves and the human societies responsible for their maintenance and spread. Genetic analyses of domesticates complement archaeobotanical and archaeological data in addressing major questions regarding the evolution and dispersal of cultivated plants and the (pre)history of agricultural origins and development. The crops that have received the most attention in this regard are those with the highest global economic importance today, including wheats , rice . Panicum miliaceum is a temperate representative of Panicum, a large and primarily pantropical genus which also includes the economically important biofuel species switchgrass .In the current study, we analyse the structuring of genetic diversity in one of the world's oldest cultivated crops, broomcorn millet  P. Beauv), broomcorn millet was the staple cereal of one of the world's independent centres of agricultural origins, in northern China. The earliest published record comes from the Yellow River valley site of Cishan, where diagnostic Panicum-type phytoliths have recently been identified in conjunction with 14C dates in the range 10 300–8700 cal bp (bp) and abundant carbonized grains from the Xinglonggou site in eastern Inner Mongolia . Analysis of the phylogeography, domestic origins and spread of P. miliaceum are, therefore, significant not just for the crop itself but for understanding the prehistory and landscape ecology of Eurasia.Along with foxtail millet . Weedy types are also found in central Europe  and a lack of sequence data, which has limited marker development. Previous analyses of variation in isozymes , AFLPs . Additional Chinese samples were collected in the field . Thermal cycling conditions were as follows: 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 60 °C for 45 s and 72 °C for 1 min, 10 cycles of 94 °C for 30 s, 53 °C for 45 s and 72 °C for 1 min and a final extension step of 72 °C for 10 min. These conditions were used for all loci except PaM023, which used an annealing temperature of 56 °C in the first 30 cycles. During the exploratory trials, variations in annealing temperature and MgCl2 concentration relative to the above protocol were also tested. PCR products were checked by electrophoresis on 1% agarose tris-borate-EDTA (TBE) gels and diluted according to concentration between 6- and 20-fold prior to analysis on an ABI3730 instrument (Applied Biosystems). Genotyping data were analysed using GeneMapper version 4.0 software (Applied Biosystems). Bins were defined for each locus, and genotypes were scored manually. Following evaluation of loci for amplification, reliability of scoring and polymorphism (see Results), the main sample set was analysed using 16 loci amplified using 15 primer pairs  was modelled using the Bayesian clustering approach implemented in the software structure version 2.3.1, using the admixture model with correlated allele frequencies, with 200 000 burnin and 1 000 000 Markov chain Monte Carlo (MCMC) reps, for 10 replicate runs of K = 1–10  reps, for 10 replicate runs of K =1–10. We calculated ΔK . The distance measure DA was employed as this has been shown to give the most reliable phylogenetic trees in analyses of microsatellite data for human populations (endroscope version 2.2 (amovas) were performed in Arlequin 3.11  was applied in order to identify stable run parameters. Other settings were left at their defaults. Convergence of chains was confirmed by rerunning analyses using the θ and M output as starting values and by inspection of chains using Tracer 1.5 (To determine the direction of gene flow (east to west vs. west to east), the 98 accessions were analysed using e 3.2.15  under boe 3.2.15 . The accacer 1.5 .P. miliaceum. For this reason, and because P. miliaceum is an allotetraploid species in which the relationship between the genomes is not known, and in which markers may potentially amplify two homeologous loci  in structure gave a minimum mean probability of −ln P(D) = 1291.9 at K=1 and a maximum mean probability of −ln P(D) = 998.72 at K=6. Evaluation of the optimum number of K following the procedure by K, at K=2 and 6. The plots of ln P(D) and ΔK against K .Bayesian modelling of the number of homogeneous genepools . As described below, these two clusters share overlapping geographic distributions and are relatively close to one another genetically. These small differences do not, therefore, have any substantial impact on the interpretation of the results. For the following analyses, we used the output from the analyses in structure.Barplots of the proportional allocation in K =6. This agreement improved when samples with a high degree of admixture (highest assignment proportion to any one genepool <0.6) under the six-genepool structure model are removed from the genetic distance analysis. The tree with admixed samples excluded is shown in The neighbour-joining tree inferred from the genetic distance matrix showed moderate agreement between the relationships indicated by the phenogram topology and those from the Bayesian clustering analysis using structure, we constructed a neighbour-joining tree of the genepools proposed by the K =6 structure model, using the modelled allele frequencies as input data with bootstrapping across loci. This tree  for the relationship of the pink and cyan clusters. amovas, in which the samples were grouped according to their majority proportional allocation in the structureQ-matrices, found significant and moderately strong genetic differentiation for both groups defined by K =2  and by K=6 .Bootstrap resampling of the neighbour-joining tree found very low support for the internal nodes. This result is typical for genetic distance trees based on relatively few markers. To look for statistical support for phylogenetic relationships between the clusters identified by K =2) divides the accessions into two clusters with strong respective eastern and western foci  cluster in the two-cluster model is divided into four genepools (K =2 model subdivides into two under K=6. One of these subdivisions (yellow cluster) forms a geographically coherent group of samples from northwest Kazakhstan and the Russian Plain and is also scattered in Central Asia and northwestern China. The other (red cluster) predominates in Ukraine and the Caucasus and is scattered through Morocco, Central Asia, western South Asia, China and Mongolia. With the exception of genepool 4 (cyan), the samples from China include at least one accession with a very high assignment proportion to each of the genepools, a mix that is not seen in any other region of the map.The model with six genepools, which is also strongly supported by the enepools , with thenepools . One cluenepools  suggestsstructure for the K=2 and 6 models are shown in K=6 grouping show similar levels of diversity in the red, yellow, green and blue clusters. The pink cluster shows somewhat higher diversity. The cyan cluster shows no diversity, with all loci monomorphic. A comparison of the red and blue clusters defined by the K =2 model found somewhat higher diversity samples allocated to the blue (eastern) than to the red (western) cluster.Diversity statistics for groups of samples with the highest proportional allocation to each of the clusters identified by migrate were inconclusive. Despite large final ESSes , values for all parameters varied greatly between runs without any obvious pattern. Estimated thetas and Ms for individual runs varied both for each marker and for the final average. Repeat analyses using the same starting parameters yielded dissimilar results. This is probably because of the small number of alleles per marker (median = 4) preventing the resolution of genealogies and directions of migration, a known issue with migrate and similar programs  group has a centre of distribution in China (bp (P. miliaceum in the European (but not as yet the Chinese) Neolithic. For example, Panicum grain size during and beyond the Neolithic period have been observed  are present within China, and the amova results indicate moderately strong genetic differentiation between these clusters. As shown in in situ from the pink cluster within Japan or possibly Korea. This separation is likely to have been relatively recent, as suggested by the lack of genetic diversity within the cyan cluster  genetic cluster identified by the bp , the most extensively investigated site of the Central Asian Neolithic . As noted by c. 20%) of accessions belonging to the red genepool are in China and Mongolia. It is possible that these represent an introduction from a western domestication of the red cluster, perhaps comigrating with other cereals introduced from the west in the 4th/5th millennia bp ] that we can be confident that this is a real pattern. Genetic diversity typically exhibits strong geographic stratification at a distance from a centre of domestication, with samples from the centre of origin itself being heterogeneous for the diversity within the crop. This is consistent with the observed distribution.Considering the genetic data, the diversity statistics indicate that the red cluster supports less diversity than the blue cluster overall. This argues against the red heartland in the west representing a centre of origin, although it should be noted that the individual subclusters (red and yellow) have similar diversity to the subclusters in the blue (eastern) group. We also note that a substantial minority ( pattern . The proamova results, showing that these two subclusters are genetically well-differentiated.We now consider the alternative major model, namely a single domestication centre in China that gave rise to all populations. The increasing dominance of the red cluster west from northern China could represent a founder effect from diverse populations in the centre of origin, paralleling the similar founder effects inferred that have led to the current distribution of the green and pink clusters . It is nThe hypothesis of a single centre of origin for broomcorn millet in China, followed by its westward expansion, would imply that this crop reached Central Asia prior to its arrival in eastern Europe. As discussed above, this entails chronological inconsistencies with the published archaeobotanical data. This emphasizes the need for increased research both on the chronology and nature of the Neolithic in Central Asia and on evidence for cultivation and use of broomcorn millet in early Neolithic Europe. It is undisputed that broomcorn millet became a significant cereal in many parts of Europe from the Bronze Age onward , but theP. miliaceum. Analyses to determine the direction of migration were uninformative for our data set. The genetic picture would be clarified by comparison of landrace genetic diversity with that of the wild ancestor of broomcorn millet. Analysis of microsatellite diversity in P. miliaceum subsp. ruderale could determine whether this subspecies is indeed the wild ancestor of the domesticated form, in which case the former would be expected to maintain a more diverse genepool, or a derived feral type, whose genetic diversity is a subset of domesticated P. miliaceum. We did not include any samples of P. miliaceum subsp. ruderale in the current study: Although morphotypes fitting the description of this taxon are reported as being widespread across the Eurasian steppe (P. miliaceum subsp. ruderale is not listed on the http://www.agroatlas.ru website, and no specimens are identified as belonging to this taxon in the extensive herbarium collection of P. miliaceum at the Royal Botanic Gardens, Kew. Appropriate field collections of weedy forms of P. miliaceum for genetic comparison with cultivated types are needed, but the necessary fieldwork across vast areas of Eurasia, to give a sample set from which reliable conclusions could be drawn, would require a major international collaborative project. Our demonstration of strong phylogeographic patterning in cultivated P. miliaceum makes fieldwork and sampling of P. miliaceum subsp. ruderale a high priority for further investigation.The archaeobotanical and genetic data thus currently present a set of signals that are not wholly consistent with either a single or multiple domestication centres for n steppe , detailebc (P. miliaceum, relationships between these traits and genome-wide diversity in this crop remain hypotheses to be tested. Whatever the underlying mechanisms, it is clear that the spread of broomcorn millet across the Eurasian steppe region has left a marked phylogenetic signature. There is growing interest in the development of agriculture in Central Asia (Evolutionary and population processes other than the primary spread from centres of domestication may have contributed to the observed patterns of genetic variation in broomcorn millet. Firstly, population movements later than the Neolithic may have played a role, e.g. along the Silk Routes which supported trade between Europe and Asia from the 1st millennium bc . The rel