In situ diversity and population genetic structure of eight cultivated sorghum landrace populations were investigated in the center of origin, Ethiopia using seven phenotypic traits and 12 highly polymorphic sorghum SSR markers. In farmers’ fields, DNA samples were collected using Whatman® plant saver card and quantitative phenotypic traits were measured from 160 individual plant samples belonging to the eight populations representing three diverse geographical regions. High diversity was observed among the various populations for the measured phenotypic traits. The 12 SSR loci produced a total of 123 alleles of which 78 (63.41%) were rare (frequency ≤0.05) with an average of 10.25 alleles per polymorphic locus. The polymorphism information content (PIC) was in the range 0.39-0.85 showing the good discriminatory power of the SSR loci used. Average observed heterozygosity and gene diversity across all populations and loci ranged 0.04-0.33 and 0.41-0.87, respectively. Neighbor-joining and STRUCTURE analyses grouped the 160 samples from the eight populations differently. AMOVA showed 54.44% of the variation to be within populations, 32.76% among populations within regions, and 12.8% among the regions of origin. There was high divergence in the total populations (FST = 0.40) indicating low level of gene flow (Nm = 0.38), but high gene flow was also observed in some adjacent populations. The populations from Wello displayed close relationship with remote Gibe and Metekel populations indicating that the variation followed human migration patterns. Implications of the results for sorghum improvement and germplasm conservation are discussed.Genetic diversity is a fundamental input for every plant breeding program, genetic resources conservation, and evolutionary studies. The online version of this article (doi:10.1186/2193-1801-3-212) contains supplementary material, which is available to authorized users. Sorghum bicolor) is the fifth-most important cereal in the world. It serves as a staple for millions of people in Africa and Asia (Ejeta and Grenier injera (common leavened flat bread) next to tef  Moench]. Ethiopian farmers grow mixed sorghum landraces of diverse forms in their fields for various local purposes. The Ethiopian sorghum germplasm has been highly contributing to the global agriculture. Singh and Axtell  investigating the genetic diversity of sorghum landraces sampled in situ from three agroclimatic regions of Ethiopia using phenotypic traits and SSR markers; 2) investigating the factors shaping the population genetic structure of sorghum landraces; and 3) suggesting measures to aid efforts of crop improvement and genetic resources conservation.Phenotypic traits may not give reliable estimate of genetic diversity as these traits are limited in number and due to environmental influence , two in Gibe river valley , and two in Metekel zone . The different landrace collections are known by different local names  (Garmin), which was later overlaid on to the regional map of Ethiopia using ArcGIS version 9.3  and sizes were read using the associated GeneMapper 3.7 software  and manually scored. To exclude the possible effects of imprecise DNA fragment sizes due to stuttering, large allele drop out, or null alleles on genotyping, the software Allelobin  in the field on seven common quantitative phenotypic traits following descriptors for sorghum  were computed using FSTAT software (Goudet s) and private allelic richness (Rp) were computed using the rarefaction method   of the total populations and pair wise FST among all-pairs of populations were computed using FSTAT software (Goudet m) as (1-FST)/4FST. Shared alleles distance matrix (Jin and Chakraborty http://taxonomy.zoology.gla.ac.uk/rod/rod.html). Further, the pattern of population structure and detection of admixture was visualized using a Bayesian model based clustering method implemented in STRUCTURE software, Version 2.2  was computed using Arlequin v 3.1 software  matrix. The geographic distance among the collection sites was computed from geographical coordinates marked with the aid of GPS using the web based Geographic Distance Matrix Generator (GDMG) version 1.2.3 software of the American Museum of Natural History, Center for Biodiversity and Conservation (http://biodiversityinformatics.amnh.org/open_source/gdmg/index.php). Later, the correlation between Rousset’s genetic distance matrix and the geographic distance matrix of the collection sites was calculated using the web based program IBDWS version 3.23 (available at http://ibdws.sdsu.edu/~ibdws/). Significance of the correlation was tested using Mantel  to 470 cm (Metekel-1) averaging 289 cm. Six of the eight populations showed an average height of greater than 3 m. Leaf width ranged from 4.4 cm (Wello-2) to 12.5 cm (Metekel-1). Leaf length was also in the range of 42 cm to 100 cm and leaf number was in the range of six (Wello-2) to 24 (Wello-4) , except between number of tillers and head length and width, and leaf length; between head length and leaf number, and between leaf length and leaf number. Plant height and leaf width had highly significant positive correlation with the remaining quantitative phenotypic traits. The first three principal component axes explained 80.53% of the total variation. Plant height contributed the largest factor loadings (0.44) for PCA1. PCA2 is mostly influenced by number of tillers per plant (0.56). For PCA3 leaf length contributed the largest share of the variation (0.40). Figure Availability of alleles in each locus  ranged from 0.93 Sb4-121) to 1.0  with mean 0.97. All of the 12 SSR loci were highly polymorphic with PIC values ranging from 0.38 to 0.85 (mean = 0.62) (Table 21 to 1.0o) was in the range of 0.05-0.32 with mean 0.13 across all loci. Gene diversity was the lowest in Gibe-1 (He = 0.20) population and the highest in Wello-2 (He = 0.70) population and its value averaged over all populations and loci was 0.67 (SD = 0.11). W2 population had also the highest allelic richness. Allelic richness and private allelic richness over all pairs of populations and loci were significant (p < 0.05). M1 population had the highest (Rp = 0.83) and G1 had the lowest private allelic richness (Rp = 0.11). Wello as a region of collection supported the highest gene diversity (He = 0.70) whereas Gibe was the lowest (He = 0.40), but values were significant between Gibe and Wello , and between Metekel and Wello . Allelic richness was the lowest in Gibe (Rs = 3.9) and the highest in Wello (Rs = 6.8), but differences were significant between Gibe and Metekel  and between Gibe and Wello , but not significant between Metekel and Wello . Similarly, private allelic richness was significant between Gibe and Metelkel  and between Gibe and Wello , but not significant between Metekel and Wello .The values of the various genetic diversity indices of the eight populations are presented in Table ST = 0.40, p < 0.001)  . The Neighbor-joining dendrogram grouped the 160 individuals of the eight populations into three major clusters  in the overall populations. However, gene flow as high as Nm = 3.66 was observed in the adjacent Metekel populations (M1 and M2).AMOVA showed 54.44% of the variation to be within populations, 32.76% among populations within regions, and 12.8% among the regions of origin  . Moreover, the reduced major axis (RMA) regression showed a significant relationship with an intercept , slope (0.003 ± SE0.0006) and with coefficient of determination . All of the populations included in the present study displayed such characters except the only improved exotic variety, 76 T1#23 which showed parameters deviated from such measurements. For instance, all except Wello-2 and Wello-3 populations exhibited average height between 300 and 360 cm. Wello-3 (Jigurte) population is relatively shorter and earlier maturing than the high yielding and previously the dominant cultivar called Degalit (Wello-4). Jigurte is now becoming the dominant cultivar in Kobo-Alamata plain due to its earlier maturity than the other landraces and its better suitability to the changing climate, mainly to unreliable rainfall in the region. Some farmers call it as “America” perhaps because it was introduced from another place decades ago. The observed high variation in the range of the quantitative phenotypic traits in all populations could have genetic basis or it could be due to phenotypic plasticity. If it is due to the latter, it could be due to the differences in the rainfall and temperature as there was little variation in altitude of the collection sites (1088-1500 m) to bring about such changes. Ayana et al.  studied The observed SSR fragment sizes were within the range of the sizes in the previously published reports in sorghum  was rather unexpected. This variety was released in 1976 and its distribution to farmers all over the country has also long history. Thus, it might be because the variety was mixed with the landraces and lost its genetic purity. As a region of collection, Gibe populations were found to have the lowest diversity and Wello populations the highest in terms of allelic richness and number of private alleles. The significantly lower genetic diversity indices of the Gibe and Metekel populations than those of the Wello populations may indicate some level of genetic drift  during sampling of the seeds by migrants during settlement. Farmers usually carry few heads when they migrate and these may represent few genotypes only. Gibe and Metekel areas had no history of sorghum production before settlement.Although there was no comparison of the present e = 0.66) was similar to Kenyan sorghum accessions (He = 0.66)      was comparable to what was observed in Djé et al. (o = 0.134) and a bit higher than in Barnaud et al. (o = 0.11), but much higher than the result of Deu et al. .u et al.  compiledex situ accessions can sometimes experience loss of variability associated with missing of low frequency alleles (<1%) during repeated regeneration , the dominant cereal, which might have resulted in genetic erosion of the sorghum landraces in these regions. The adoption of early maturing improved varieties was also found to be high in two of such areas affected by recurrent drought, Kobo in the North Eastern and Mieso in the Eastern parts of the country .In some areas like the extreme North Wello  once up on a time sorghum was the dominant crop and highly diverse. At present, however, a et al.  proved lhttp://www.ibc.gov.et/biodiversity/conservation/database-ms). This number is very small compared to the germplasm preserved elsewhere. For instance, more than 7000 germplasm accessions of Ethiopian origin are preserved at the US National Plant Germplasm System  reached 9432  for over 15 years. Currently, he is working for Advanta Seed International, a UPL Group company as a sorghum breeder for the African continent based in Ethiopia.Additional file 1: Table S1: Characteristics of the SSR primers  used in the study. (DOCX 14 KB)Additional file 2: Table S2: Number of individuals from each pre-determined population assigned by STRUCTURE to the two clusters (k=1 and k=2) with ≥90% probability of membership. (DOCX 11 KB)


Eucyclogobius newberryi), show that reductions in both factors typically coincide, and describe how low levels of diversity contribute to the extinction risk of these isolated populations. We illustrate that, for this annual species, continuous reproduction is a safeguard against reproductive failure by any one population segment, as natural, stochastically driven salinity increases frequently result in high mortality among juvenile individuals. Several study populations deviated from the natural pattern of year-round reproduction typical for the species, rendering those with severely truncated reproductive periods vulnerable to extinction in the event of environmental fluctuation. In contrast, demographically diverse populations are more likely to persist through such periods through the continuous presence of adults with broader physiological tolerance to abrupt salinity changes. Notably, we found a significant correlation between genetic diversity and demographic variation in the study populations, which could be the result of population stressors that restrict both of these diversity measures simultaneously, or suggestive of a causative relationship between these population characteristics. These findings demonstrate the importance of biocomplexity at the population level, and assert that the maintenance of diversity contributes to population resilience and conservation of this endangered species.Theoretical work has shown that reduced phenotypic heterogeneity leads to population instability and can increase extinction potential, yet few examples exist of natural populations that illustrate how varying levels expressed diversity may influence population persistence, particularly during periods of stochastic environmental fluctuation. In this study, we assess levels of expressed variation and genetic diversity among demographically independent populations of tidewater goby ( Life-history diversity and genetic variation are two forms of biodiversity deemed worthy of conservation by the World Conservation Union (IUCN), and are considered essential to retaining fitness and long-term adaptive potential of populations Eucyclogobius newberryi), a short-lived, endangered fish that inhabits dynamic coastal habitats subject to frequent environmental changes within geographically isolated populations in the context of the species’ biology, and suggest that reduced levels of variation, particularly in the temporal extent of the reproductive period, render affected populations more susceptible to localized extirpation during periods of environmental disturbance.In this study, we investigated age composition and genetic diversity among isolated populations of tidewater goby , annual fish endemic to California, that occurs in brackish, isolated, and often very small (less than 10 hectares) estuaries along the coast, from just south of the Oregon border to San Diego, USA A number of life history adaptations allow tidewater goby populations to persist in these environmentally challenging habitats. Most notably, reproduction occurs throughout the year, though increased spawning activity is generally observed during summer and fall In this study, (1) we used a controlled experiment to confirm that salinity increases associated with breaching causes mortality to the early life stages, and (2) show how continuous reproduction (as measured by population age composition) can buffer isolated populations of tidewater goby against adverse impacts  resulting from these events. Further, (3) we assessed the genetic diversity of 17 populations of this species in northern California, and (4) investigated relationship between genetic diversity measures and demographic heterogeneity after accounting for habitat size (as a proxy of habitat heterogeneity). We discuss the results in the context of the biology of this endangered fish species, and argue that resilience to environmental stochasticity increases when life history variability and healthy levels of genetic variation are retained in a population.All work was conducted under California Scientific Collecting Permit SC-10527 and in concordance with institutional protocol at Humboldt State University , where all samples have been deposited in the scientific fish collection .To evaluate size-specific mortality associated with salinity increases experienced by tidewater goby populations during a breaching event, we exposed two size groups of tidewater goby  captured in BL . We used Welch’s two sample T-test to test for significant differences in survival between size groups.We captured tidewater goby using a 3.3 m by 1.3 m seine with a 1.58 mm mesh in water no deeper than the height of the seine and no shallower than 0.2 m. As neither our field observation nor any literature report indicate age- or size-specific segregation of tidewater goby, we considered collections representative subsamples of the respective goby populations.We obtained estimates of genetic diversity and corresponding individual length measurements  for eleven populations from a previous study (sampled in 2006) t , it is improbable that many individuals of different ages reach identical, or similar, sizes at time t . Following this reasoning, we consider the length range of samples an approximation of the variation in ages within populations of this short-lived fish. However, to further validate that size measurements provide a nonlethal alternative to direct  age estimation, we conducted a more detailed age and growth study on two large populations using otolith analysis (described in the following section).In organisms with indeterminate growth, particularly those with a short lifespan, the size distribution of individuals is conservatively reflective of the population’s age composition. In other words, while individuals of the same age may obtain different sizes at time HE] and allelic richness [rA]) through linear regression analysis. Alternatively, we used the standard deviation (SD) in lengths observed within populations to investigate the relationship between demographic variation and genetic diversity, as the observed length range may be strongly affected by presence of rare individuals with atypical (extreme) sizes.We evaluated the relationship between the length ranges observed in our study populations  and their genetic diversity (mean Hardy-Weinberg expected heterozygosity [10hectares) to control for effects of environmental differences among the isolated habitats in a general linear model fit.As the size of the habitat to which a population is constrained may influence the duration of the reproductive period and limit populations size , we used area  for comparison. In Big Lagoon (BL), we collected individuals from April 2009 until August 2010, and sampled the second location, Arcata Marsh (AM), from April 2009 until October 2011. We measured all individuals in the field to the nearest millimeter , and sacrificed and preserved a subsample of 20–25 fish spanning the length range of goby encountered . Concurrent with field sample collection, we obtained salinity measurements  using a VEE GEE STX-3 refractometer.Unlike most animal taxa, fishes can often be aged directly through analysis of periodically deposited increments in bony structures, most notably their otoliths (earstones). We used otolith microstructural analysis to estimate the ages (in days) for large numbers of individuals from two populations in northern California, USA, for age determination . We began enumeration of daily increments at the first continuous increment outside the core region, and counted increments continuously to the otolith margin. We discarded the sample when continuous enumeration not possible on either of the two otoliths.We extracted the sagittal otoliths, rinsed them in water and ethanol and mounted them on a microscope slide using wet’n’wild “Wild Shine” Clear Nail Protector (type 401A). After curing, we polished the otoliths by hand using waterproof sandpaper  and 6 µm lapping film  to reveal daily increments. We counted daily increments under a compound microscope under 500X total magnification, using an oil-immersion lens (MEIJI Model #10824), and captured images captured with a Lumt is age (in days), a is the inverse of time, and b is a dimensionless constant. We then reconstructed birthdate distributions for these two populations by tabulating, for each fish, the estimated daily age subtracted from its collection date.To validate daily growth increment deposition, we induced chemical marks in otoliths by immersing tidewater goby in a 5% calcein solution. We marked a total of 1441 tidewater goby and released them into their natal environment BL, , and usemple see ,  and  tp<0.001). No mortality was observed in control groups of either size class. Surviving fish were released into their natal environment.Mean mortality across 11 replicates of the “small” category was 0.50 (SD = 0.10), and 0.0 across the 5 replicates of the “large” category , illustrp<0.001 ANOVA, Length-frequency distributions were highly variable among populations, with size ranges and mean sizes ranging from 8 mm to 32 mm and 20.0 to 41.4 mm, respectively. Considering that the maximum size of tidewater goby encountered during our surveys was 55 mm TL, these differences among population are substantial. Populations separated by a distance of less than four kilometers and collected within a one-week time span exhibited different mean lengths (HE) ranged from 0.08 to 0.57, while rarefied allelic richness (rA) ranged from 1.36 to 4.92  after accounting for the effects of habitat area in a general liner model analysis (model: range(mm)/SD = log10(hectares)+HE). Similarly, allelic richness explained a significant proportion of age/size variation (model: range(mm)/SD = log10(hectares)+rA; p = 0.026 and 0.020). Though explaining some of the variation, the effect of habitat area was not significant in any of the four models (p>0.2), and this variable was excluded from the model fits for illustration purposes. The univariate relationships between these two measures of genetic diversity and two indicators of demographic diversity in goby populations are shown in We detected a correlation between the temporal extent of the reproductive season and genetic diversity. A significant proportion of the variation in demography (as measured by observed size ranges and SD) was explained by expected heterozygosity . Daily age estimates ranged from 26 to 363 days for gobies sampled from the AM and from 48 to 421 days for the BL population. Estimates of instantaneous mortality rate of tidewater goby from the BL and AM populations correspond to 2.4% and 1.32% annual survivorship Back calculation of birth dates for individuals from the AM population indicated a truncated reproductive period that manifests itself as a narrow size distribution of individuals ; Table 1The buffering effects of biological diversity on ecosystem processes, likened to the portfolio effect from economic theory, are frequently invoked as an important argument for the conservation of biodiversity The results presented herein suggest that this portfolio effect not only applies to species which occur as distinct segments in a stock complex, but also to the resilience of individual, isolated populations. For tidewater goby, continuous reproduction independent of season (and the resulting diverse population age composition) appears to act as a natural safeguard against localized extinction by spreading the risk of high juvenile mortality across many different population segments. The implication of a truncated single spawning period for long-term population persistence was illustrated during a temporary increase in salinity at the Arcata Marsh (AM), a natural yet stochastic process in coastal habitats of tidewater goby. The timing of this disturbance coincided with a period of population turnover, shortly after the brief reproductive period but before juveniles could grow to sufficient size to better tolerate elevated salinity levels. Juvenile individuals, comprising over 94% of the population, vanished as a consequence of this disturbance . As lengA similar salinity increase, but at a different location , resulted in a stark decrease in abundance of smaller individuals  is an important component of the species’ biology. Consequently, the occurrence of breaching should not cause excessive stress on populations are characterized by a diverse age composition despite high juvenile mortality; rather, breaching threatens population persistence only if the population is composed of individuals of similar age, and if it occurs at a time when the great majority of individuals are of small size (shortly after peak reproduction). Further, the importance of breaching events as a population stressor may differ across the species’ range: previous work suggests that populations in the southern portion of the species’ range are subject to different risk factors related to a much drier local climate, such as hypersaline conditions or drying up of the estuary during the summer months t, Our surveys of other tidewater goby in northern California not only indicate that heterogeneity in reproductive timing appears to be severely constrained in some of the remaining populations, but also suggest that such reductions in biocomplexity are often observed in populations with low levels of genetic variation . It shouIf reproductive timing in tidewater goby is under genetic influence, the striking asynchrony in reproductive timing in adjacent populations may be explained by the random loss of genetic variation through genetic drift. Though we interpret the results presented herein as suggestive of genetic impoverishment being a causative factor leading to decreased within-population heterogeneity, we recognize that alternative explanations for the observed patter should be considered. Specifically, a reduction in birthdate variation owing to periodic failure in recruitment or size selective mortality due to unsuitable environmental conditions may explain a limited range in the size of individuals and have caused erosion in genetic variation within those populations. However, as recruitment failures occur stochastically and not periodically, we believe that the observed pattern of demographic homogeneity in populations with low genetic diversity, considered in the context of the species’ life history characteristics and their applicability to the dynamic environments in which the tidewater goby occurs, indicates that limited variation is reproductive timing is attributable to reductions in genetic diversity within isolated tidewater goby populations. Irrespective of whether a causative effect exists between genetic- and demographic diversity, limited age variation within these populations renders them particularly susceptible to environmental disturbance during certain times of the year through elimination of stabilizing effects provided by within-population variability.We do not propose that near-term extinction of extant tidewater goby populations is accurately predicted by measured heterozygosity levels, and while stochastic events, likely in the form of natural salinity increases, will probably trigger future localized extinctions of tidewater goby in the species’ northern range, we believe that this study clearly illustrates that demographically complex populations of this annual species are more resilient to such stressors. Likewise, though environmental factors doubtlessly have some influence on observed length ranges within isolated populations, the highly significant relationship between this measure of life history- and genetic diversity implies that the level of genetic variation is related to demographic heterogeneity. Though a causative relationship may be subject to debate, at a minimum, our results illustrate that a lack of genetic diversity works synergistically with environmental disturbance to influence population extirpation. As such, these results not only affirm the notion that genotypic and phenotypic diversity are tightly linked and deserve equal consideration in the quest for species conservation, but also illustrate the applicability of buffering portfolio effects to isolated populations, which contribute to their resilience against stochastic disturbances.Our findings suggest that environmental instability during and shortly after periods of peak reproductive activity is of particular concern to the conservation of tidewater goby populations, especially those characterized by low levels of age- and genetic diversity. As a consequence, artificial breaching of estuaries during spring and summer  should be avoided when possible. In addition, cohort assessment  should be incorporated in future surveys of tidewater goby populations, as this may permit identification of goby populations at elevated risk of near-term extirpation. Lastly, the relationship between genetic diversity and observed age variation should be investigated further, for example by experimental supplementation of genetic diversity in isolated populations through transfers of individuals from nearby, genetically diverse populations. Subsequently, frequent demographic assessments could be used then to evaluate whether reproductive variability has increased as a result of genetic supplementation, which may then receive consideration as a management tool in the quest for conservation of this endangered species.Figure S1Schnute’s (1981) four-parameter growth curves for populations of tidewater goby from Big Lagoon (BL), CA, and the Arcata Marsh (AM), CA.(TIFF)Click here for additional data file.Figure S2Length frequency composition of tidewater goby from Big Lagoon, CA, sampled monthly between April 2009 and August 2010.(TIFF)Click here for additional data file.Figure S3Length frequency histogram of the Big Lagoon, CA, and Arcata Marsh, CA, tidewater goby populations prior and subsequent to a stochastic, temporary increase in salinity.(TIFF)Click here for additional data file.File S1Multilocus genotypes for tidewater goby, Eucyclogobius newberryi, from 17 populations in northern California. Information on relevant primers and amplification protocol can be found in the main text.(TXT)Click here for additional data file.


Osmerus mordax) is composed of two glacial races first identified through the spatial distribution of two distinct mtDNA lineages. Contemporary breeding populations of smelt in the St. Lawrence estuary comprise contrasting mixtures of both lineages, suggesting that the two races came into secondary contact in this estuary. The overall objective of this study was to assess the role of intraspecific genetic admixture in the morphological diversification of the estuarine rainbow smelt population complex. The morphology of mixed-ancestry populations varied as a function of the relative contribution of the two races to estuarine populations, supporting the hypothesis of genetic admixture. Populations comprising both ancestral mtDNA races did not exhibit intermediate morphologies relative to pure populations but rather exhibited many traits that exceeded the parental trait values, consistent with the hypothesis of transgressive segregation. Evidence for genetic admixture at the level of the nuclear gene pool, however, provided only partial support for this hypothesis. Variation at nuclear AFLP markers revealed clear evidence of the two corresponding mtDNA glacial races. The admixture of the two races at the nuclear level is only pronounced in mixed-ancestry populations dominated by one of the mtDNA lineages, the same populations showing the greatest degree of morphological diversification and population structure. In contrast, mixed-ancestry populations dominated by the alternate mtDNA lineage showed little evidence of introgression of the nuclear genome, little morphological diversification and little contemporary population genetic structure. These results only partially support the hypothesis of transgressive segregation and may be the result of the differential effects of natural selection acting on admixed genomes from different sources.The North-east American Rainbow smelt ( Numerous intraspecific phylogeographic studies show how many vertebrate species are composed of morphologically and genetically distinct lineages resulting from the vicariant isolation of intraspecific groups in isolated refugia, particularly those created during Pleistocene continental glaciation events . HistoriHybridization may also have positive effects on fitness, including heterosis (hybrid vigor) at the individual level and/or high genetic variance for relevant phenotypic traits at the population level , 10. TheStudies of morphological traits in some hybrid populations have reported the presence of extreme phenotypes with trait values exceeding the range of parental trait values, a phenomenon known as transgressive segregation , 15. ThiOsmerus mordax), a widespread, abundant, carnivorous fish of northeastern North America occupying lakes, rivers, estuaries and coastal waters, provides an opportunity to assess the consequences of genetic admixture on morphological divergence and population structure. Anadromous/estuarine ecotypes naturally occur from the upper St. Lawrence River to Newfoundland and southwards along the Atlantic seaboard [The rainbow smelt . All fish collection sites were located on public lands and their locations are specified in To assign each fish of the 15 anadromous populations to its historical race and thus establish the extent of mixing of the two races at each sampling site, we identified diagnostic mtDNA lineages according to . Three eTo quantify phenotypic diversity, we measured 36 continuous morphometric traits and seven discrete meristic traits , all conWe applied a statistical procedure employing Principal Component Analysis, Discriminant Function Analysis and ANOVA models to quantify the nature, extent and sources of variation in phenotypic diversity. We first log transformed all morphometric data to approach multivariate normality. We then standardized morphometric measures for fish of different sizes as we are concerned principally with differences in ecologically relevant morphological traits independent of the size effect. We performed a Principal Components Analysis (PCA) , 32 withWe first tested if the morphology of mixed-ancestry populations varied as a function of the relative contribution of the two races using a multivariate regression model. Because the variables were not multivariate normally distributed, exact MANOVAs based on permutation tests were used. As the regression was highly significant (see below), we formed 2 groups of mixed populations, one dominated by the Acadian race  and a second dominated by the Atlantic race  occurring in populations characterized by the absence or presence of mixed ancestry  using a nearest neighbor nonparametric discriminant function analysis (DFA). We employed 3 contrasts to test several hypotheses. First, we hypothesized that the morphology of groups composed of populations derived from each historical race and showing no evidence of genetic mixture are divergent, as demonstrated in lacustrine populations by Barrette et al. . Secondlormation  with a Bormation .We quantified, with random ANOVA models, the relative importance of the major among- and within-population sources of variation acting to differentiate groups according to the canonical axes. The random ANOVA model provides a quantitative means to partition the variation observed on each canonical axis among the designated groups. The variance components were estimated by the maximum likelihood method  via the P< 0.0167 following the Bonferonni correction for multiple comparisons.In the case of a significant effect of genetic mixture, median values of traits significantly correlated with the discriminant functions were compared for evidence of transgressive segregation. Medians and their variance were used as the morphological trait values were not normally distributed. Traits of mixed-ancestry populations were considered as being transgressive if their medians were significantly smaller or larger than the smallest and largest median trait values, respectively, of populations showing no evidence of mixed ancestry of historical lineages . We restSmelt of different age classes were sampled at different seasons during 2011–2012 throughout the St. Lawrence River SLR; , Table 2Three categories of fish were sampled. Firstly, reproductive fish were sampled on known spawning grounds to examine the degree of reproductive isolation among putative local populations. Secondly, we sampled adult smelt captured during the winter recreational ice fishery and, thirdly, young-of-the-year (YOY) fish, at several sites , Table 2EcoRI/MseI primer pairs in selective PCRs (ATG/CAG and ACC/CAC). Fragments were migrated on ABI capillary sequencer and peaks with a minimum relative fluorescence of 100 units were scored manually using GeneMapper 3.7. For a subset of 153 individuals scored for AFLPs (We extracted DNA from EtOH-preserved tissues with the QIAGEN blood and tissues extraction kit (QIAGEN), and quantified DNA concentration by spectrophotometry. We generated AFLP fragments for 447 individuals following the AFLP Plant Mapping protocol . We used ca. 100 ng of DNA for the restriction-ligation step and two or AFLPs , we sequstructure 2.3.3 [K = 1–20). We used the criteria of Pritchard et al. [et al. [K). Figs. were made with distruct [ST between sample sites were estimated based on the distance matrix of pairwise differences between AFLP profiles with arlequin 3.5 . Finally, an analysis of molecular variance (AMOVA) was conducted to assess if clusters account for a significant proportion of genetic variance beyond that occurring among samples within clusters. Calculations were done using 1000 permutations in arlequin 3.5. [We used re 2.3.3 , 41, 42 d et al.  and Evan [et al.  to inferdistruct . The mosquin 3.5 45. FiK = 2. First, we assessed the extent of genome admixture of the founding nuclear races using two independent methods. Namely, we used q, the Bayesian coefficient of ancestry provided by structure for K = 2, and h, the maximum-likelihood hybrid index estimated with introgress [structure, we considered samples from Montreal and du Loup River as representative of the reference and alternative parental populations for nuclear DNA, respectively.To assess the patterns of admixture between the two founding races, we considered the results of structure with trogress , 47. Forq) in cluster 2 (when K = 2) and, in a second model, as a function of h, the maximum likelihood hybrid index. The Hosmer-Lemeshow test was used to confirm the goodness of fit of both models to the data. To quantify the concordance between the nuclear and mtDNA races, the area under the receiver operating characteristics (ROC) curve was calculated.Finally, we tested the correspondence between the nuclear and mtDNA evidence for two ancestral races using a logistic regression analysis , where tFWilk’s lambda = 21.43; df = 42, 314; P< 0.0001). When grouping estuarine smelt populations characterized by the extent of mixture of the two historical mtDNA races , the morphologies of the 4 groups differed significantly . All three discriminant functions were significant (P< 0.0001), with the first function explaining 58.7% of the variance, and the second and the third functions explaining 26.6% and 14.7% respectively. Reclassification of smelt to their group of origin revealed that smelt from populations of mixed ancestry were more successfully reassigned  than smelt from populations of pure ancestry . The suite of correlated traits accounting for the greatest degree of morphological divergence discriminated smelt from Atlantic-mixed populations (P = 0.0019). No significant differences occurred among the remaining 3 population groups. Contrasting Atlantic-mixed populations with pure Atlantic populations explained 47.8% of the variance associated with this function  whereas no significant differences occurred among the remaining 3 groups. Contrasting Acadian-mixed populations with pure Acadian populations explained 52.9% of the variance associated with this function (The second suite of correlated traits (function 2), discriminated smelt from Acadian-mixed populations . This pofunction . Contrasfunction . BrieflyOf the 22 morphological traits contributing significantly to the first discriminant axis and the 15 contributing to the second axis, 15 and 9, respectively, exhibited median trait values that were either significantly smaller or larger in mixed ancestry populations when compared with populations with no evidence of mixed ancestry .ST values were highly significant (see structure (see below) explained a large proportion of the total genetic variance , almost 5 times more than that accounted for by variance among samples within groups .Among the 447 smelt collected in the SLR and successfully analyzed, a total of 108 AFLP bands were unambiguously scored, of which 64 were polymorphic. However, 35 of these polymorphisms were uninformative, occurring at a frequency lower than 5% in any one sample. We thus retained 29 bands for subsequent analyses. Although only 29 bands were retained, we considered this adequate for population definition. The associated Fcant see  and the structure using Pritchard’s criterion supported the definition of K = 7 distinct genetic clusters along the SLR ,  the SLR  and 5, t the SLR  and 5, t the SLR  and 5, t the SLR  and 5 an the SLR  and 5. T the SLR  and 5, inificant ; 19 of tK = 2 distinct genetic clusters  indicate that admixture occurred principally upstream and along the north shore of the SLR with little evidence of admixture occurring along the south shore (h) and the coefficient of ancestry   conforms to the expectation of a genetically admixed population (as it was so stocked). Similarly, the North Shore Population , and the Fjord Population  are also admixed relative to all other populations.Estimates of the degree of genetic admixture between the two races (th shore . The hybc Prior)  decline oc Prior . Loc Priq or h . In the first and second models, respectively, 70.9% and 67.7% of the predicted probabilities were in concordance with observed assignments. Moreover, variables q and h are highly correlated to each other (Pearson correlation r = 0.89) and the inferred ancestry (q) is the most important of the two variables for classifying individuals. After considering the effect of q, the index h explains no further part of variation . The blue cluster illustrated in Finally, the logistic regression models revealed that the probability of being classified in the mtDNA Atlantic race increases significantly with The morphology of mixed-ancestry populations (co-occurrence of Acadian and Atlantic mtDNA races) varied as a function of the relative contribution of the two races to estuarine populations, supporting the hypothesis of genetic admixture as opposed to ancestral polymorphism. If the lineage composition of these populations were uniquely due to the retention of ancestral polymorphisms, there would be no reason to expect that populations dominated by one or the other lineage would be consistently morphologically distinct relative to populations comprising a single lineage. Pure estuarine populations of the two races are morphologically distinct, as observed in lacustrine populations . We obseThe observations presented above are consistent with the hypothesis of transgressive segregation. Evidence for genetic admixture at the level of the nuclear gene pool, however, provides only partial support for this hypothesis. The nuclear genome of rainbow smelt revealed clear evidence that two races were admixed within some populations of the purported secondary contact zone within the St. Lawrence system. In particular, the admixture of the two nuclear races is pronounced in mixed-ancestry populations dominated by the Atlantic race, the same populations showing the greatest degree of morphological differentiation. Earlier studies of the St. Lawrence sympatric smelt populations identified head length, eye area and jaw length as being responsible for the strong morphological differentiation between the ‘north-shore’ and ‘south-shore’ St. Lawrence estuary populations , 28. RelCoincidentally, many of the populations comprising the St. Lawrence smelt complex reside in the fluvial estuary, along the north shore of the middle estuary and in the Saguenay fjord, areas associated with the greatest degree of admixture of the 2 nuclear gene pools. This may be largely due to the strong physical heterogeneity of the region, favoring reproductive isolation through philopatry. These areas and the south shore of the estuary are only separated by several tens of kilometers, well within the dispersal capacity of smelt . AtlantiThe pattern of admixture based on nuclear DNA did not always reflect the pattern of admixture suggested by mtDNA. The Atlantic mtDNA lineage composed approximately 20% of the mtDNA gene pool of the South Shore and Chaleur Bay populations, but these populations showed little evidence of introgression of the two nuclear gene pools and differed morphologically from pure Acadian populations to a far lesser degree. As a result, the correspondence between nuclear and mtDNA races, albeit significant, is not perfect. Although we cannot discount completely the retention of ancestral polymorphism as a contributing factor, the lack of complete concordance between nuclear and mtDNA races most likely reflects introgression of mtDNA lineages between the two ancestral nuclear DNA races.Gasterosteus aculeatus) that evolved under greater seasonal temperature extremes following invasion of lakes from the sea [The lack of evidence for admixture of the two nuclear DNA races along the south shore, in spite of evidence for the likely introgression of the mtDNA races in this area, may involve active selection against the Atlantic genome. In contrast to the north shore of the SLR, the smelt sampled in different spawning tributaries along the south shore of the SLR shows little evidence of introgression of the nuclear genome and no contemporary population genetic structure. Furthermore, the Acadian-mixed populations occupying the St. Lawrence and the estuaries of New Brunswick exhibit little morphological diversification relative to populations comprising uniquely the Acadian lineage. As selection is typically expected to favor locally adapted genotypes and can act against admixed individuals, there may be some conditions under which admixture will have negative impacts on population fitness . Althoug the sea . If we sOur findings are not entirely consistent with the biogeographical hypothesis of two routes of postglacial re-colonization of North-east North America by smelt . The preCoregonus spp. [We set out to find evidence that genetic admixture within rainbow smelt populations contributed to the morphological diversification and population structure of the species in northeastern North America rather than to a net loss of diversity through genetic homogenization and an averaging of morphological features. We have presented two contrasting outcomes for the role of natural selection within the smelt population complex, acting both for and against admixed genomes. Other evolutionary processes, not documented in this study, are no doubt involved in the evolution of this species complex. Nevertheless, the presence of extreme phenotypes associated with admixed populations of Atlantic origin implicates transgressive segregation in the diversification of the estuarine population complex. It has been proposed that genetic admixture among the cichlid fishes of the East African rift lakes may increase evolvability by providing new phenotypic variation previously unseen by selection, through transgressive segregation , 59. Hisnus spp. . The hypnus spp. , 61 may S1 Protocol(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Ne), is greatly influenced by human activities. In this context, wild pathogen populations can provide valuable information for assessing the long‐term risk associated with crop pests. In this study, we estimated the effective population size of the beet cyst nematode, Heterodera schachtii, by sampling 34 populations infecting the sea beet Beta vulgaris spp. maritima twice within a one‐year period. Only 20 populations produced enough generations to analyze the variation in allele frequencies, with the remaining populations showing a high mortality rate of the host plant after only 1 year. The 20 analyzed populations showed surprisingly low effective population sizes, with most having Ne close to 85 individuals. We attribute these low values to the variation in population size through time, systematic inbreeding, and unbalanced sex‐ratios. Our results suggest that H. schachtii has low evolutionary potential in natural environments. Pest control strategies in which populations on crops mimic wild populations may help prevent parasite adaptation to host resistance.The sustainability of modern agriculture relies on strategies that can control the ability of pathogens to overcome chemicals or genetic resistances through natural selection. This evolutionary potential, which depends partly on effective population size ( Ne) is a key evolutionary parameter that corresponds to the size of an idealized population  subjected to the same amount of genetic drift as the population of interest. Effective population size thus informs about the strength of genetic drift within populations and depends on demographic factors such as census population size, dispersal ability, or the mating system. It is considered as a fundamental factor in the estimation of the risk associated with resistance in parasite populations  and is one of the most damaging pathogens of sugar beet crops , the wild ancestor of all cultivated beets, also is one of the hosts of H. schachtii in natural ecosystems  expectations suggest extensive inbreeding due to mating between relatives , Granville Nord , Granville Sud , and Saint Léonard , were separated by distances ranging from 300 m to 150 km  and manual examination of filtrates. Cysts were stored at 4°C in moistened sand before molecular characterization.Soil samples were collected around roots of  km Fig. . Populat km Fig. . Thirty‐ km Fig. . In the Heterodera species; therefore, molecular characterization based on the restriction profiles of ITS sequence was used for species identification. By multiplying the ratio of H. schachtii among the 40 larvae with the number of cysts we sampled, we estimated the number of H. schachtii cysts contained in our samples. DNA extraction, PCR amplification of ITS sequence, and digestion of PCR products were performed as described in Gracianne et al. , and allele sizes were identified using the automatic calling and binning procedure of GeneMapper v4.1 (Applied Biosystems™), with any irregular results evaluated manually. Samples with dubious genotypes were reamplified.We ultimately genotyped 761 and 854 y et al. . MicrosaFIS, and its statistical significance, for each population. The genetic diversity of nematode populations was evaluated by estimating expected heterozygosity (He) and allelic richness (Ar) using Fstat. Allelic richness was estimated using the rarefaction method described in El Mousadik and Petit (Single and multilocus departures from Hardy–Weinberg (HW) equilibrium were tested for all populations on each beach using permutation tests (10 000 permutations) adjusted for multiple tests with Bonferroni corrections, as implemented in the software Fstat version 2.9.3 Goudet . This prnd Petit .H. schachtii in wild populations. However, H. schachtii produces about four generations a year in cultivated fields  between the temporal samples to perform this test.Both methods assume neither migration nor selection and that the variation in allele frequencies between temporal samples is due only to genetic drift and sampling errors. To verify this hypothesis, we performed an exact test of homogeneity in allele frequencies between temporal samples to detect significant changes in allele frequencies using the software Genepop v4.2 . Overall, adding up all of the generations over the entire year resulted in 9.9 generations for Montfarville beach and 10.6 for the three other surveyed beaches. Wild H. schachtii populations may thus have produced up to 10 generations between the two sampling sessions. The number of generations that elapsed between the two sampling sessions was therefore set to the minimum (four generations), maximum (10 generations) and their median values (seven generations) in subsequent analyses.The modeled relationship between temperature and the minimum generation time of P = 0.67) for finite populations. Effective population sizes obtained when considering a ten‐generation span were twice as large as results with a four‐generation span .The exact homogeneity tests detected significant changes in allele frequencies between the two sampling sessions in nine populations. Of these, only population two exhibited a significant action of nonrandom forces such as selection or migration .In the case of FIS estimates can give further information on the likelihood that new generations were found in the 2013 samples. The life cycle of H. schachtii and the low dispersal ability of larvae result in inbreeding and/or substructuring (Wahlund effect), which leads to an increase of heterozygote deficiencies over time . Furthermore, the number of cysts observed in our samples (corrected by the proportion of H. schachtii cysts relative to other species) was remarkably similar between finite and infinite populations , with a mean number of cysts 158.2 ± 19.8 (SEM) and 175 ± 23.8, respectively, refuting the hypothesis that infinite population size results from huge census sizes.The analysis of temporal variation in mean multilocus Ne estimates remained infinite when we decreased the number of generations from ten to four. Overall, our findings suggest that infinite population sizes correspond to nematode populations lacking significant temporal variations in allele frequencies, probably because new nematode generations were not produced between the two sampling sessions.In addition, infinite estimate of effective population size also may be caused by an overestimation of the number of generations between samples, which reduces the effect of genetic drift. However, all infinite Ne was overestimated for this population. Therefore, we considered that our populations were not affected strongly by migration or selection during the one‐year interval between the two samples and that the assumptions of the temporal method held for the finite‐size populations, thereby giving reliable estimates.Temporal methods assume no significant influence of other micro‐evolutionary forces, such as migration or selection. The relevance of this assumption in our dataset was tested by applying a generalized test developed by Waples : only poH. schachtii per 100 g of soil. Assuming that each cyst contains between 500 and 600 eggs . For comparison, estimates of the effective population size of the free‐living nematode Caenorhabditis elegans range (i) from 200 to 9600 individuals with AFLP markers . Low effective sizes have been observed in other plant pathogens such as fungi  rates suggest that these two evolutionary forces are too weak to enhance the evolutionary potential of these populations at the host scale. Finally, this calls into question the capacity of this nematode to adapt to changing environments unless selection intensity is strong  or tolerant beet varieties . Given that our results have shown that H. schachtii populations are naturally vulnerable to genetic drift without anthropogenic influence, clever use of this feature could prevent the parasite from overcoming plant resistance. Crop rotation is an efficient way to modify population census size of H. schachtii through time, by introducing large variation in host availability, similar to that experienced in wild populations. This method was once considered as difficult to implement for control of H. schachtii: this nematode is a generalist able to parasitize most Brassicaceae species. Crop and weed species that can be used to reduce the census size of H. schachtii are now known, leading to the development of nematicidal intercrops  and maximum number of generations of Heterodera schachtii produced every month between the two sampling sessions (lines).Click here for additional data file.Table S1. Waples generalized test results for populations with significant temporal changes in allelic frequencies (exact homogeneity test).Click here for additional data file.


Parrotia subaequalis (Hamamelidaceae) is a Tertiary relic species endemic in eastern China. We used inter‐simple sequence repeat (ISSR) markers to access genetic diversity and population genetic structure in natural five populations of P. subaequalis. The levels of genetic diversity were higher at species level (H = 0.2031) but lower at population level (H = 0.1096). The higher genetic diversity at species levels might be attributed to the accumulation of distinctive genotypes which adapted to the different habitats after Quaternary glaciations. Meanwhile, founder effects on the early stage, and subsequent bottleneck of population regeneration due to its biological characteristics, environmental features, and human activities, seemed to explain the low population levels of genetic diversity. The hierarchical AMOVA revealed high levels (42.60%) of among‐population genetic differentiation, which was in congruence with the high levels of Nei's genetic differentiation index (GST = 0.4629) and limited gene flow (Nm = 0.5801) among the studied populations. Mantel test showed a significant isolation‐by‐distance, indicating that geographic isolation has a significant effect on genetic structure in this species. Unweighted pair‐group method with arithmetic average clustering, PCoA, and Bayesian analyses uniformly recovered groups that matched the geographical distribution of this species. In particular, our results suggest that Yangtze River has served as a natural barrier to gene flow between populations occurred on both riversides. Concerning the management of P. subaequalis, the high genetic differentiation among populations indicates that preserving all five natural populations in situ and collecting enough individuals from these populations for ex situ conservation are necessary. Parrotia subaequalis  based on a single fruiting specimen collected from the Longchi Mountain, Jiangsu Province of China (Chang Shaniodendron (tribe Fothergilleae), was proposed to accommodate this taxon  in the China Red Data Book, with a very narrow distribution range. The five known relict populations of P. subaequalis comprise no more than 100 reproductive individuals. Therefore, this is a species with a high conservation priority. To date, previous studies about P. subaequalis mainly focused on its phylogenetic placement, taxonomic status, morphology, distribution ranges, habitat ranges, and ecophysiological traits  markers were employed to assess the genetic diversity and population genetic structure in natural populations of P. subaequalis were collected throughout its entire distribution region. Two populations (AWL and AWX) are distributed in Wanfo Mountain of Anhui Province, two additional populations (JLS and JLF) occur in Longchi Mountain of Jiangsu Province, and the fifth population (ZLM) is found in Longwang Mountain of Zhejiang Province 2SO4 [50 mmol/L], EDTA [0.1 mmol/L], Thesit [50%]), glycerol, MgCl2 [2.5 mmol/L]), 0.2 mmol/L each of dATP, dCTP, dGTP and dTTP, 1.5 unit of Taq DNA polymerase , 0.5 mmol/L random 10‐base primer, and 40 ng genomic DNA. The PCR amplifications were run for 45 cycles at 94°C for 1 min (denaturation step), 50°C for 45 sec , and 72°C for 5 min (elongation step), preceded by an initial melting step of 5 min at 94°C, and followed by a final extension step of 5 min at 72°C.Genomic DNA was extracted according to the modified CTAB method  were screened for amplification, ten of which  were selected for PCR amplification because they exhibited clear bands, were polymorphic, and showed reproducibility. As negative controls, we performed PCRs containing all the PCR components except DNA template.N  were removed from data analyses to avoid bias in parameter estimations (Lynch and Milligan Amplification products were separated by means of electrophoresis on 1.5 percent (w/v) agarose gels in 1× TAE buffer . Sizes of the amplification products were estimated by comparisons with a standard molecular weight marker . Amplified fragments were scored as presence (1) or absence (0) of homologous bands, and were then transformed into a binary matrix. Bands with a frequency of < 3/H) (Nei I), Nei's unbiased genetic distance (ND) (Nei NI) (Nei HT), gene diversity within populations (HS), Nei's genetic differentiation index among populations (GST) (Nei Nm). An estimate of Nm (number of migrants per generation) value among populations was computed using the formula of Nm = 0.5(1 − GST)/GST  or absent (0) for each locus. This resulted in a data matrix that was the basis for subsequent analyses. We used the program POPGENE version 1.32  Nei , ShannonND) Nei , Nei's uNI) Nei , total gST) Nei , and genThe significance of correlation between Nei's unbiased genetic distances and geographic distances (in km) was performed by Mantel test Mantel  with 999The obtained genetic distance matrix was then used to perform a cluster analysis [based on the unweighted pair‐group method with arithmetic average (UPGMA)] using MEGA 5.0  was performed to partition the total genetic variance among populations and among geographic regions (either south of the Yangtze River or north of Yangtze River), using the AMOVA program 1.55  of runs. Finally, the clustered output was visualized using the software Distruct version 1.1  of these loci were polymorphic among the sampled populations. At the population level, the mean values of the Nei's gene diversity, Shannon information index, and the percentage of polymorphic loci were 0.1096, 0.1698, and 36.11%, respectively. The JLF population  in Longchi Mountain had the highest genetic diversity, while the ZLM population  in Longwang Mountain had the lowest genetic diversity. At the species level, the Nei's gene diversity, Shannon information index, and the percentage of polymorphic loci were 0.2031, 0.3132, and 68.52%, respectively  was primarily distributed among populations (HS = 0.1096 ± 0.0110). The value of Nei's genetic differentiation index among populations (GST) was 0.4629, indicated that a relatively high level of genetic differentiation existed among the five populations. The hierarchical AMOVA revealed that 42.60% of the total variation was attributed to differences among five populations and that 57.40% was contributed by differences within populations (P < 0.001)  was 0.5801.The total gene diversity , and north of Yangtze River . The AMOVA revealed that 27.80% of the total variation was distributed among geographic regions, whereas 72.20% was found within geographic regions (P < 0.001)  Table .NI = 0.9154) existed between populations JLS and JLF in the Longchi Mountain (Table NI = 0.8031)  generated three axes with interpretable patterns Fig. . The firk value with the highest value of ΔK for the 139 individuals was K = 2       , and AMOVA (42.6%) values indicated significant genetic differentiation among the studied populations. The Gst values observed for P. subaequalis are above the averages observed for endangered and endemic plants with same life‐history traits    and Schisandra sphenanthera (Magnoliaceae)  pattern in P. subaequalis genetic resources involves not only preventing extinction but also ensuring the availability of resources for the changing environments in the future. Currently, all known populations are protected , it is desirable to keep ex situ collections represent the north and south populations of the Yangtze River and far apart populations separate, to prevent unwanted crosses among these populations in the botanical gardens (Zhang et al. Our study represents only part of a comprehensive conservation research plan that may lead to the long‐term survival of the species. Other research needed includes a long‐term population monitoring and dynamic study as well as the species's habitat requirement, which will lead to better management measures that guarantee the long‐term viability of the species.None declared.


Halophila ovalis and its closely-related species belonging to the Hydrocharitaceae are well-known as a complex taxonomic challenge mainly due to their high morphological plasticity. The relationship of genetic differentiation and geographic barriers of H. ovalis radiation was not much studied in this region. Are there misidentifications between H. ovalis and its closely related species? Does any taxonomic uncertainty among different populations of H. ovalis persist? Is there any genetic differentiation among populations in the Western Pacific and the Eastern Indian Ocean, which are separated by the Thai-Malay peninsula? Genetic markers can be used to characterize and identify individuals or species and will be used to answer these questions.The Indo-Pacific region has the largest number of seagrass species worldwide and this region is considered as the origin of the Hydrocharitaceae. H. ovalis belonged to the H. major clade, also supported by morphological data. Evolutionary divergence between the two clades is between 0.033 and 0.038, much higher than the evolutionary divergence among H. ovalis populations. Eight haplotypes were found; none of the haplotypes from the Western Pacific is found in India and vice versa. Analysis of genetic diversity based on microsatellite analysis revealed that the genetic diversity in the Western Pacific is higher than in the Eastern Indian Ocean. The unrooted neighbor-joining tree among 14 populations from the Western Pacific and the Eastern Indian Ocean showed six groups. The Mantel test results revealed a significant correlation between genetic and geographic distances among populations. Results from band-based and allele frequency-based approaches from Amplified Fragment Length Polymorphism showed that all samples collected from both sides of the Thai-Malay peninsula were clustered into two clades: Gulf of Thailand and Andaman Sea.Phylogenetic analyses of the nuclear ribosomal internal transcribed spacer region based on materials collected from 17 populations in the Western Pacific and the Eastern Indian Ocean showed that some specimens identified as H. major for Malaysia and Myanmar. The study also revealed that the Thai-Malay peninsula is a geographic barrier between H. ovalis populations in the Western Pacific and the Eastern Indian Ocean.Our study documented the new records of  Halophila members is still unclear and genetic variation has not been much investigated so far[Halophila ovalis (R. Br.) Hooker is the dominant species and very commonly found in the region. Recently, some new records of Halophila members such as Halophila major (Zoll.) Miquel, were documented in Southeast Asian countries including Indonesia, Viet Nam and Thailand[H. sulawesii J. Kuo was found and described for the first time in Indonesia[H. ovalis and closely related species based on leaf morphological data is very challenging, and species misidentification among Halophila members is reported in various studies[The Indo-Pacific Ocean – the origin of seagrass - has the largest number of seagrass species worldwide with huge meadows of mixed species stands, but the taxonomy of d so far. In compThailand,3. Additndonesia. Traditi studies,5,6. Gen studies.The Indo-Pacific Ocean also shows a high diversity of landscapes, habitats as well as several existing geographic barriers. Geographic isolation refers to a situation where a species, or a population of a species, becomes separated by a physical barrier, allowing each group to diverge along separate evolutionary paths. The effHalophila, H. ovalis is widespread in the Indo-Pacific Ocean. In the Pacific, it occurs from southern Japan throughout Southeast Asia, many islands of the western Pacific, and through all but the southern coast of Australia, as well as Lord Howe and Norfolk Islands, and as far east as Tonga and Samoa. In the Indian Ocean, H. ovalis is found from southwestern Australia to East Africa and the Red Sea, including Madagascar, with the exception of islands or coastlines with no records. Recently, H. ovalis has been also discovered in the Atlantic Ocean on the Island of Antigua[Halophila ovalis grows on a variety of substrates and is often the first to colonize newly available sediments[H. ovalis reproduces vegetatively by branching of rhizomes and the formation of new shoots, and sexually through seeds[Among the members of  Antigua. The pla Antigua. Halophiediments,17. The ediments,18. Thisediments. Like otgh seeds. Due to gh seeds emphasizHalophila species[Halophila genus[H. ovalis belonged to different clades, and this clearly points out the need for critical taxonomic revision of Halophila material from the entire geographic distribution of this genus[H. ovalis and closely related species namely H. major, Halophila nipponica J. Kuo, Halophila minor (Zoll.) den Hartog and Halophila hawaiana Doty and B. C. Stone[Leaf morphology is used as the main key to identify and name  species,20. Howe species. Recentlla genus. Howeveris genus. This nuC. Stone,7,22,23.. Microsatellites are simple sequence repeats (SSRs) with advantages like locus-specificity, co-dominance, high degree of polymorphism, and it is also possible to work with partially degraded DNA[H. ovalis.There are several techniques including isozyme analyses,25, Randaded DNA. So far H. ovalis persists and (ii) geographic distance, differentiation of habitats or the geographic barrier of the Indo-Malay peninsula may affect the genetic variation of H. ovalis from the Western Pacific to the Eastern Indian Ocean. The aims of this study are (i) to identify Halophila species collected in Hong Kong, Thailand, Malaysia and India based on the molecular marker (ITS1-5.8S-ITS2) and (ii) to search for the genetic structure of H. ovalis from the Western Pacific to the Eastern Indian Ocean based on microsatellite and AFLP approaches.It is hypothesized that (i) taxonomic uncertainty among different populations of Halophila spp. in the study sites shown in Figure H. major (clade I) and H. ovalis (clade II) with 98, 100, 96 and 99% bootstrap values, respectively. There was no difference in the topology of the phylogenetic trees based on these different methods except for small differences in the bootstrap values. In clade I, haplotypes (Hap.) 4, 5, 13, and 16 clustered with known H. major sequences. In clade II, the remaining haplotypes clustered with known sequences from H. ovalis. None of the samples clustered with known sequences from H. minor  need to be grouped into the H. ovalis clade instead with H. minor sequences. For both countries, Malaysia and Myanmar, it is the first time that H. major was recorded.Nineteen ITS sequences  and lamina margin (R) showed clear differences in comparison to H. ovalis. The ratio of the distance between the intra-marginal vein and the lamina margin was 1:20.8 to 1:25.6. In contrast, this ratio was 1:12 to 1:16 in H. ovalis[H. major was 18 to 20 and therefore higher than the number of paired cross veins in H. ovalis (14 to 17)[r/R was higher than 0.05 (homoscedasticity). Single factor ANOVA shows that for the ratio (r/R) significant differences can be observed among the collection sites . Details resulting from multiple comparisons of each trait obtained by the Tukey test showed that there were significant differences of the ratio (r/R) between populations at MY-mb, MY-gs, MM-gt (H. major) and the remaining populations . Details of comparisons of the leaf morphology of H. major and H. ovalis are presented in Table The morphological data also supported the results obtained from the molecular ITS data. For the samples identified as H. ovalis. Moreove ovalis 1 to 17[4o), expected heterozygosity (He) and allelic richness (A) are presented in Table e) or genetic diversity in the Western Pacific and the Indian Ocean were found at MY-jo and TH-sa, respectively. The lowest expected genetic diversity in the Western Pacific and Indian Ocean were observed at HK-tc and IN-ma, respectively. Genetic diversity of the populations in the Western Pacific Ocean was slightly higher than of the populations in the Indian Ocean (0.306 vs 0.289). However, there was no significant difference between the oceanic systems . Likewise, observed heterozygosity and allelic richness in the Western Pacific were slightly higher than in the Indian Ocean . For the observed heterozygosity and allelic richness, there was no statistically significant difference between both oceanic systems .Data on observed heterozygosity , among populations within groups (p < 0.01) and within populations (p < 0.01)  Table . Hence, H. ovalis populations in the two regions VII and IX  showed significant differences among groups (p < 0.01), among populations within groups (p < 0.01), and within populations (p < 0.01). The overall genetic variation from the Halophila populations in the Eastern Indian Ocean was 0.485 calculated from FSTAT.For the Eastern Indian Ocean, a very high genetic distance between the two regions VII and IX, ranking from 0.731 to 1.296 was observed Table . For theST = 0.679). The correlation between geographic and genetic distances in the study area is presented in Figure Mantel < 0.0001, the significance level α = 0.05). An approximately linear increase in Slatkin’s genetic distance with increasing geographic distance between all pairs of populations confirmed a simple model, namely differentiation-by-distance.The unrooted neighbor-joining tree among 14 populations from eight regions in the Western Pacific and Eastern Indian Ocean showed six main groups including group 1 - Region I: Northern part of the South China Sea (HK), group 2 - Region II and V: Western part of the South China Sea and the Gulf of Thailand , group 3 – Region III and IV: Eastern part of the South China Sea and the Celebes Sea , group 4 – Region 6: Southern part of the South China Sea (MY-jo), group 5 – Region VII: Andaman Sea (TH-tr and TH-sa) and group 6 – Region IX: Bay of Bengal (IN-ma and IN-ka) . Haplotype I  commonly occurred in the South China Sea, Celebes Sea and Andaman Sea. Haplotypes II (Hap. 7) and III (Hap. 8) were found in MY-bd and MY-gm (Celebes Sea), respectively, and haplotype IV (Hap. 10) in MY-jo only. In the Andaman Sea, there was one more haplotype present – haplotype V (Hap. 15). Three haplotypes  that did not occur in the South China Sea, Celebes Sea and Andaman Sea were found in the Bay of Bengal. Haplotype VI (Hap. 19) was identified in IN-ka, while haplotypes VII (Hap. 17) and VIII (Hap. 18) were detected in IN-ma  and within the Gulf of Thailand (TH-kn) were 0.565 to 0.928 and 0.624 to 0.822, respectively. The similarity values between the populations of TH-tr and TH-sa were higher than between the population of TH-tr and TH-kn (0.634 to 0.820 vs 0.582 to 0.731).For the AFLP analysis, the genetic similarities (Dice index) among the 24 individual H. ovalis populations were divided into two groups, either collected in the Gulf of Thailand or in the Andaman Sea . However, results of clustering individuals of TH-tr and TH-sa were not significant. The plot of a principal coordinate analysis (PCoA), based on individual genetic distances calculated with 208 AFLP markers, is presented in Additional fileH. ovalis was clearly distributed in two main clades: Gulf of Thailand clade and Andaman Sea clade.The cluster analysis Figure  revealed. Gene diversity (H)[ST[ST) among populations of H. ovalis revealed that the genetic distance between TH-tr and TH-sa populations  was lower than between TH-tr and TH-kn populations . The dendrogram based on Nei’s genetic distance also showed that three populations were divided into two main clades: 1) Gulf of Thailand and 2) Andaman Sea .Based on the above results, the entire samples were organized in two groups: Gulf of Thailand and Andaman Sea groupsity (H) of the ey (H)[ST was 0.19H. ovalis collected from the Western Pacific Ocean to the Eastern Indian Ocean using nuclear sequence comparison (ITS) and two DNA fingerprinting approaches: AFLP and SSRs. Conformation of new records for H. major in Malaysia and Myanmar and detection of high levels of polymorphism underlined impressively that genetic markers are powerful tools for species identification and assessing genetic diversity in seagrass.The present study is the first report of genetic diversity, as well as genetic differences, within and among populations of Halophila genus, namely H. ovalis[H. hawaiana[H. nipponica[H. major was unclear, because of overlapping leaf characteristics between H. ovalis and H. major. Molecular markers, especially ITS, were shown to be a valuable tool in resolving genetic relationships among the species of Halophila. For instance, Halophila euphlebia Makino was once treated as synonym for H. ovalis[H. major[H. major and H. ovalis are distinct species based on ITS analysis and morphological data. Recently, Short et al.[Halophila currently reported in Myanmar and Malaysia, respectively,[H. major. Halophila major has been found in the recent years along the coastlines of Southeast Asian countries including Indonesia, Thailand, Viet Nam and Japan[Halophila members could not be fully resolved among closely related species such as H. ovalis, H. major and H. ovata Gaudich based on concatenated sequences of the two plastid markers rbcL and matK[H. ovalis, H. major and H. minor are distinct species[H. ovalis and H. major revealed that the materials collected in Mabul Island (MY-mb) and Gusungan Island (MY-gs), both in Malaysia, and one population (MM-gy) in Myanmar differ significantly from the H. ovalis clade. Moreover, four methods of constructing phylogenic trees also indicated that materials collected in Mabul Island and Gusungan Island  and Myanmar are H. major. Only the indications of the molecular methods initiated a detailed microscopic analysis of the leaf samples. The leaf morphology based on the ratio of the distance between the intra-marginal vein and the lamina margin confirmed the ITS analysis. Hence, morphological and nuclear sequence (ITS) analysis indicated that the materials collected in Mabul Island and Gusungan Island  and one population in Myanmar are actually H. major. In the field this kind of analysis is usually not possible, but our results suggest that careful analysis of seagrass samples need to be conducted before classifying them as H. ovalis.Variation of leaf morphology has been detected within several species of the H. ovalis,51, H. h hawaiana, and H. nipponica. Short enipponica argued tH. ovalis,53; then[H. major. Results[H. major supportectively,,54 not ind Japan. As it wand matK,21. In c species,23. HencHalophila species, summing up to seven species[H. ovalis haplotypes: there are four haplotypes found in six populations in Malaysia. In contrast, Nguyen et al.[The Indo-Pacific region has the largest number of seagrass species worldwide and this region was considered as the origin of the Hydrocharitaceae family,55. Mala species, this stn et al. found onZ. marina (0.504 to 0.601)[Zostera noltii Hornemann (0.442 – 0.630)[Posidonia oceanica (L.) Delile (0.191 to 0.363)[Cymodocea nodosa (U.) Ascherson (0.286 to 0.564)[H. ovalis is lower. Unfortunately, there are no studies on the genetic diversity of H. ovalis based on SSRs markers for direct comparison so far. In other AFLP approaches of seagrass species the genetic diversity such as Thalassia testudinum Banks ex König , (0.310 o 0.601), Zostera– 0.630), Posidono 0.363) and Cymoo 0.564),  is sligo 0.072,). Hence,ST = 0.483), among populations in the Eastern Indian Ocean (FST = 0.485), and larger significant differences among 14 populations surveyed in the Western Pacific Ocean and the Eastern Indian Ocean (FST = 0.679). All above results indicated that great genetic differentiation among populations was detected and/or gene flow among populations is very low. For the Celebes Sea, pair wise genetic differentiation among populations showed genetic differentiation although the geographic distance among populations is about 30 to 40 km. However, genetic differentiation between MY-mg and MY-bd is very low (0.048) in contrast to other populations in the Celebes Sea. This could be explained by the diversity of the habitat such as substratum, currents, and time exposure to air during low tide etc., and those factors may affect the genetic differentiation. Japar et al.[H. ovalis, which grows in different substratum and depth. Significant genetic differences were also found in Z. marina between the Wadden Sea and the Baltic Sea where geographic distance among populations is within areas of 10 to 50 km[H. ovalis collected in Tiga Island showed great differences in comparison to the other populations in the Celebes Sea .The present distribution of the genetic structure within species is influenced by evolutionary history. In thisr et al. stated tto 50 km. Leaf moH. ovalis populations in the open sea and the lagoon were also found in India based on AFLP data[For the South China Sea, there are very great genetic differentiations among populations in the northern part of the South China Sea (HK-tc) and the remaining populations in the western, eastern and southern part of the South China Sea. Perhaps high latitude (or lower average temperature) in the northern part of the South China Sea may lead to the genetic differentiation. Both populations collected in Viet Nam also showed significant differences, although the geographic distance between two populations is less than 100 km. In fact, there are great differences between the environmental conditions from two populations, in the lagoon and in the open sea. It could be explained by the differentiation of salinity, with high salinity (open sea) and low salinity (lagoon). The genetic difference between FLP data. For theFLP data shows suThe genetic differentiation between populations in the Andaman Sea and in the Bay of Bengal is significantly different. This could be explained by a very long geographic distance  between the Bay of Bengal and the Andaman Sea. However, there is no significant genetic differentiation between populations of TH-tr and TH-sa in the Andaman Sea. Perhaps short geographic distances and the same habitat are the main causes that led to the high similarity between the two populations. Results from AFLP analysis also indicated that the genetic distance between populations from TH-tr and TH-sa within the Andaman Sea is much lower than between populations from the Andaman Sea and the Gulf of Thailand. Moreover, surface currents in the winter (from TH-tr to TH-sa) and in the summer (TH-sa to TH-tr) support H. ovalis populations in the study. However, one of the most striking results is the unexpected result in the case of the HK-tc population. It showed no simple relationship between genetic differentiation and distance between pairs of population. The HK-tc population was genetically closer to the population in the Eastern Indian Ocean than to populations in the Western Pacific Ocean. At present we are unable to explain this puzzling result.The result from the unrooted neighbor-joining tree based on Slatkin’s genetic distance showed the identified six main clusters corresponding to populations from different regions. Based on the genetic distance, the population in MY-jo seems to be in between the Western Pacific and the Eastern Indian Ocean, which corresponds to the geographic distribution of H. ovalis – H. ovata complex. High level of polymorphic bands has previously been reported in Thalassia testudinum Banks ex König[Zostera marina Linnaeus[H. ovalis populations found in India[ST) and genetic distance  were polymorphic bands. This contrasts with a level of variability of 30% using AFLP in land plant species, such as rice. In a reex König and ZostLinnaeus using AFin India. Compari animals,13 and m animals,15 also H. ovalis, results indicated that the genetic markers are powerful tools to assess the genetic differentiation on the broad sample collection sites. However, the sample size was still low and in the case of TH-kn that may affect the standard error of the diversity in the population of the species as discussed by Singh et al.[Based on ITS, AFLP and SSRs analysis of genetic variation of h et al.. AccordiH. major for Malaysia and Myanmar. The study also revealed that the Thai-Malay peninsula is a geographic barrier of H. ovalis populations in the Western Pacific and the Eastern Indian Ocean. Characteristics of habitat are also an ecological barrier to the evolution of H. ovalis in the smaller scale area.Our study documented the new records of Halophila species were carried out at the Pacific Ocean and the Indian Ocean. Samples were collected from 17 populations belonging to eight regions depending on the geographic distribution. Regions were determined by long geographic distance  or geographic barrier. Region I (northern part of South China Sea): 1-Hong Kong (HK-tc). Region II (western part of South China Sea): 2-Van Phong (VN-vp), 3-Thuy Trieu (VN-tt). Region III (eastern part of South China Sea): 4-Sarawak (MY-sr). Region IV (Celebes Sea): 5-Tiga Island (MY-tg), 6-Mabul Island (MY-mb), 7-Gusungan Island (MY-gs), 8-Sibangat Island (MY-sb), 9-Bodgaya Island (MY-bd), 10-Maiga Island (MY-mg). Region V (Gulf of Thailand): 11-Kanom (TH-kn). Region VI (southern part of South China Sea): 12-Johore (MY-jo). All above six regions belong to the Pacific Ocean. Region VII (eastern part of Andaman Sea): 13-Satun (TH-sa), 14-Trang (TH-tr). Region VIII (northern part of Andaman Sea): 15-Myanmar (MM-gy). Region IX : 16-Marakanam (IN-ma), 17-Kanyakumari (IN-ka). Details of each sampling site are presented in Figure Samplings of r) and lamina margin (R) were measured under the microscope Olympus SZ . Photographs were taken using a U-TV1X-2 digital camera (Olympus) connected to a computer. The test for equal variances of each data set of leaf morphology among groups was checked by Levene’s test for homoscedasticity. Levene’s test, one-way analysis of variance (ANOVA), Tukey test was carried out by Minitab software . Specimens were identified using the keys of Kuo et al.[For the morphological analysis, ten adult leaves collected from ten different individuals from each location were used for the analysis. The five most important and differentiating parameters of leaf morphology including lamina width, lamina length, number of paired cross veins, space between intra-marginal veins and the ratio of the distance between intra-marginal vein , 10 to 30 ng template DNA, 1 pmol primer each. The PCR was performed in a PTC 200 thermocycler  with a heated lid under the following conditions: initial denaturation for 4 min at 95°C followed by 30 cycles of denaturation for 25 s at 95°C, primer annealing for 30 s at 52°C and extension for 35 s at 72°C, terminated by a final hold at 10°C. All PCR reactions were repeated two to four times independently with the same individual to reduce errors, possibly created by the Taq polymerase, in the final consensus sequence to a minimum. Direct sequencing of PCR products was done by GATC Biotech  from both directions. Consensus sequence was achieved by Clone Manager 9 .In this analysis, three individuals per population randomly selected from 15 populations  described above were used for ITS amplification Table  to ampliTaq polymerase (MBI Fermentas), and 1 pmol primer each. The PCR was performed in a PTC 200 thermocycler  was added. Reactions were heated up to 72°C for 5 min before loading onto 6% AFLP gels . For running an AFLP gel on the 4300 DNA Analyzer  manufacture’s instruction were followed. Base pair lengths obtained from visual analysis was resolved with previously published allele lengths[One hundred individuals  buffer  for overnight at 37°C. Adapters were prepared in a total volume of 5 μl including 50 pmol of MseI adapters, 5 pmol of EcoRI adapters, 0.5 mM ATP and 1.2 U of T4 DNA ligase, and 1x RL buffer. The mix of digested DNA and adapters were incubated at 37°C for 3.5 h and then used as a template for PCR. The pre-selective PCR contained 5 μl of template, 1 U of Taq polymerase , 0.25 mM of each of the four dNTPs, 1x Williams buffer  and 50 ng of EcoRI and MseI primers with one selective nucleotide (A) in a total volume of 50 μl. The PCR program consisted of twenty cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C, followed by 10 min at 72°C. An aliquot of the reaction mix was diluted 1:20 with 1x TE Buffer . The selective PCR contained 2.5 μl of the diluted (1:20) product of the pre-selective PCR, 2 mM dNTPs, and 5 U Taq polymerase in a total volume of 10 μl. Four primer pairs, EcoRI + ACA/MseI + ATC, EcoRI + ACC/MseI + ATC, EcoRI + ACA/MseI + ACA and EcoRI + ACC/MseI + ACA,  were used for the selective amplification. The first amplification cycle was carried out for 30 s at 94°C, 30 s at 65°C and 1 min at 72°C. In each of the following 11 cycles, the annealing temperature was reduced by 0.7°C. The last 24 cycles were carried out at an annealing temperature of 56°C, and the final extension step was carried out at 72°C for 10 min. To each sample, 50 μl of dye (see above) was added. Running conditions and instruments were the same as for SSRs.Details of sample size, name of locations and coordinates are presented in Table Halophila decipiens Ostenfeld (KC175913) and H. minor  were aligned by CLUSTAL X[Halophila decipiens was used as out-group, because it is closer to its ancestor than the Halophila ovalis complex[H. ovalis based on ITS analysis were used for AFLP and SSRs analysis.The obtained ITS sequences and known sequence of LUSTAL X and the enfeld KC75913 andLUSTAL X was usedLUSTAL X with theLUSTAL X, and BA LUSTAL X. Halophi complex. Moreove complex. Only poi2, where pi is the frequency of the ith allele[ST[ST was calculated using the formula GST = (HT- HS)/HT[T represents the total gene diversity and HS represents the gene diversity within populations. Those values and the dendrograms (UPGMA) were assessed by POPGENE 3.2[For the AFLP analysis, only polymorphic fragments were scored as binary data . The binary scores were manually compared with the pictures to re-confirm presence or absence of bands. A presence/absence binomial matrix of 30 individuals and 201 polymorphic loci was used as basis for the analysis. In this study, the analysis with two approaches including band-based approach  and allele frequency-based approach (for population level) was carrh allele. Allelicllele[ST was usedT- HS)/HT, where HGENE 3.2 and MEGAGENE 3.2. In addiGENE 3.2). The anGENE 3.2.e) = (Σ expected frequency of heterozygotes at each locus)/; observed heterozygosity (Ho) = (Σ frequency of heterozygotes at each locus)/; and allele richness (A) = /. All those parameters were assessed by Microsatellite Toolkit for Excel[ST) was calculated. FST measures the degree of inbreeding in the subpopulation relative to the total population, and is commonly used to estimate population differentiation. The software FSTAT version 2.9.3.1[ST), among populations within groups (FSC) and within population (FCT) were test by AMOVA . This analysis was carried out by Arlequin 3.5[. Pairwise distances were calculated from allele frequency data using the Slatkin’s distance[. The unrooted neighbor joining tree was constructed using neighbor joining with bootstrap resampling  in package Phylip version 3.5[. The tree was edited and displayed in MEGA5.2[. Geographic distances (km) among populations were determined from NOAA digital map  and their Genbank number (KF620337-KF620355).Click here for filePrincipal Coordinate Analysis (PCoA) based on 208 AFLP markers. There are two groups including the Gulf of Thailand and the Andaman Sea. Clustering of TH-tr and TH-sa is not significant. Abbreviations as in Figure Click here for fileDendrogram of genetic distances among three populations of H. ovalis. Branch lengths were calculated by Nei[d by Nei. Abbrevid by Nei, edited d by Nei.Click here for file