Genetic diversity and morphological differentiation in Liatris helleri (Asteraceae), a threatened plant species

Liatris helleri (Asteraceae) is an insect-pollinated herbaceous perennial endemic to several high-elevation sites in the Blue Ridge Mountains of North Carolina. Allozymes were used to describe the genetic diversity and population structure in nine populations of this rare, federally listed species. Differences in leaf morphology were also examined for greenhouse-grown plants representing several populations. The proportion of the total genetic diversity found among populations, as indicated by the allozyme data, was 16%. Higher levels of population differentiation were found for differences in leaf shape; population of origin accounted for 37% of the variation in maximum leaf width, while families within populations accounted for 7%. In contrast to many endemic species, L. helleri maintains fairly high levels of genetic diversity. For the species, the percent polymorphic loci was 87.5, the average number of alleles at variable loci was 3.00 and the gene diversity was 0.276. Mean population values were percent polymorphic loci =58.4, mean number of alleles per polymorphic locus =2.59 and gene diversity =0.219. The estimated gene flow was low (Nm=0.85–1.32) and a relatively high correlation (r=0.55; p<0.005) was found between linear geographic and genetic distance. This suggests that the populations are partially isolated by distance, despite the limited range (<60 km) of the species. We recommend that population distinetiveness be maintained in restoration efforts.     

Genetic and clonal diversity in Korean populations ofVitex rotundifolia (Verbenaceae)

Vitex rotundifolia L.f. is a woody perennial and has sexual and asexual modes of reproduction. Allozyme study was conducted on 550 plants in 13 Korean populations. The levels of genetic variability and divergence within and among populations, respectively, are considerably lower and higher than the mean values for woody plants with similar life history tralts. Mean percentage of polymorphic loci (P _P), mean number of alleles per locus (A _P), and mean genetic diversity (He _P) within populations ofV. rotundifolia were: 16.7%, 1.21, and 0.047. On average, about 79% of the total variation inV. rotundifolia was common to all populations (meanG _ST=0.208). In addition, significant differences in allele frequencies among populations were found in all polymorphic loci examined (P<0.001). On the other hand, levels of genotypic diversity within and among populations were moderate. About 44% (18/41) of multilocus genotypes were “local genotypes” (genotypes occurring in only one population), whereas only one “widespread genotype” (genotypes occurring in more than 75% of the populations) were detected. The mean number of multilocus genotypes per population (G) and mean genotypic diversity index (D _G) were 8.4 and 0.74, respectively. Most common multilocus genotypes found in populations were homozygous for five polymorphic loci. The abundance of ramets of these genets is responsible for the low levels of expected heterozygosity within populations. The results indicate that clonal reproduction may act as an enhancer of genetic drift by reducing effective size of local populations ofV. rotundifolia.     

LOCAL GENETIC AND CLONAL STRUCTURE IN THE TROPICAL TERRESTRIAL BROMELIAD,AECHMEA MAGDALENAE

The genetic structure of populations of the clone-forming tropical terrestrial bromeliad, Aechmea magdalenae, was examined by electrophoretic analyses in nine populations occurring on and near Barro Colorado Island, Republic of Panama. For the nine populations as a whole, 33% of the loci were polymorphic and the genetic diversity was 0.121. Within populations, the mean percent of polymorphic loci was 24.1% and the mean genetic diversity was 0.084. About one-third of the total allozyme diversity resided among populations (mean G_ST = 0.356). The distribution of A. magdalenae was patchy in the study area. This, coupled with the relatively small effective population sizes and the possible founding of populations by few individuals may have contributed to the large among-population component of genetic diversity. The extent of clonal growth was inferred by examining the relationship between the proportion of rosette-pairs sharing identical multilocus genotypes and distance between pairs. This method indicates that clonal spread is local; individuals sharing multilocus genotypes most often occur within 10 m of each other.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

GENETIC DIVERSITY AND CLONAL STRUCTURE IN A COLUMNAR CACTUS,LOPHOCEREUS SCHOTTII

In southern Arizona the columnar cactus, Lophocereus schottii, inhabits desert riparian environments. Reproduction in this part of its range is predominantly asexual and occurs by either the dispersal of stems in the immediate vicinity of parents or the long-distance transport of detached stem pieces downstream by floodwaters. Genetic diversity and clonal structure of eight populations of L. schottii in Organ Pipe Cactus National Monument, Arizona, were examined. In all populations ramets were mapped and stem tissue from each ramet was examined electrophoretically. At the species level, 44.4% of the loci were polymorphic and the genetic diversity was 0.145. Within populations, the mean proportion of polymorphic loci and genetic diversity were 34.4% and 0.126%, respectively. Although most of the allozyme variation was within populations, appreciable heterogeneity was found among populations (G_ST = 0.130). Genetic and genotypic diversity was greatest in three of the four populations located in the principal area of L. schottii occurrence in the monument. Genotypic diversity was lowest in the smallest population and in the most isolated population. Ramets in all populations were spatially aggregated. Plant pairs with identical multilocus genotypes were usually ≤ 10 m apart, but some widely separated individuals had identical genotypes. Occasional long-distance dispersal of stems and the periodic recruitment of seedlings have caused genets to intermingle, promoting outcrossing and maintaining genetic diversity.     

Extremely Low Levels of Allozyme Variation in Southern Korean Populations of the Two Rare and Endangered Lithophytic or Epiphytic <Emphasis Type="Italic">Bulbophyllum Drymoglossum</Emphasis> and <Emphasis Type="Italic">Sarcanthus Scolopendrifolius</Emphasis> (Orchidaceae): Implications for Conservation

Allozyme variation was investigated in two local populations of Bulbophyllum drymoglossum and three populations Sarcanthus scolopendrifolius, two rare and endangered lithophytes and epiphytes from South Korea. Genetic diversity was extremely low within populations (mean H _e = 0.011 for B. drymoglossum; 0.002 for S. scolopendrifolius). Among the putative screened 21 loci, we found only one polymorphic locus for each species. Only one polymorphic locus, detected just one population of each species, revealed significantly high degree of population differentiation between and among populations (F _ST = 0.253 for B. drymoglossum and F _ST = 0.899 for S. scolopendrifolius). These results suggest that genetic drift (consequence of a very small effective population size), coupled with a limited gene flow would play a major role in shaping population genetics of these species in South Korea. The current status of both species (small population sizes, spatially isolated populations, and highly localized habitats) in addition to the extremely low levels of genetic diversity and reckless collection of endangered orchids by plant sellers, significantly threaten the long-term survival of these species in Korea. Conservation of the two species requires both in situ strategies, by introducing of genets to increase effective population sizes by minimizing adverse effects (e.g., outbreeding depression and genetic swamping by non-native genotypes), and ex situ strategies, such as collection of genets from clonal ramets.     

Low genetic differentiation among populations ofArabis serrata (Brassicaceae) along an altitudinal gradient

We investigated the extent of genetic differentiation among populations of fujihatazao,Arabis serrata along an altitudinal gradient at Mt. Fuji in Shizuoka Prefecture. This species is a perennial plant, widely distributed in Japan forming small isolated populations. However, at Mt. Fuji, this species constitutes a large population distributed from 1440 to 2400 m altitude. A total of 411 individuals were sampled from ten subpopulations. Eighteen loci were detected on eleven enzyme systems. Eleven loci were monomorphic and seven loci were polymorphic with a mean of 2.11 alleles per loci. Nei's genetic distance (mean 0.01) and genetic identity (mean 0.968) were very similar among populations indicating a low genetic differentiation. The total genetic diversity (H _T ) estimated for the polymorphic loci was, in average, 0.396. The mean gene differentiation (G_ST=0.091) was very low. Gene frequency of seven polymorphic loci was analyzed by spatial autocorrelation methods based on Moran's indexes. Only Pgi-3 exhibited a significant negative autocorrelation (−0.160;P<0.05); other loci values ranged from −0.134 to 0.027. Gene flow estimated by indirect methods varied between genes but most of the values were high (meanNm=20.8) suggesting that subpopulations at different altitudes are probably connected. Despite plants at different altitudes present different ecological traits (e.g., differences in phenology, growth and reproductive traits), subpopulations ofA. serrata are still low differentiated, at least for the loci studied. This may be explained by the recent origin of some habitats (e.g., second crater and surrounded areas) in this locality.     

RAPD and Allozyme Analysis of Genetic Diversity in Panax ginseng C.A. Meyer and P. quinquefolius L.

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.     

GENETIC DIVERSITY AND POPULATION STRUCTURE IN TRADESCANTIA HIRSUTICAULIS (COMMELINACEAE)

Tradescantia hirsuticaulis, the hairy-stemmed spiderwort, is an insect-pollinated perennial plant species found primarily on rock outcrops in Georgia, South Carolina, and Alabama. Although populations of T. hirsuticaulis are rare and scattered, local populations are frequently large. Levels of genetic variation were assessed for 13 populations representing the species' range in these three states. Despite the disjunct distribution of this habitat specialist and apparent lack of specialized seed and pollen dispersal mechanisms, exceptionally high levels of genetic variation are maintained within the species, with a moderate level of variation (18%) found among populations. Twenty-nine of the 33 loci resolved (88%) were polymorphic within the species; the mean number of loci polymorphic within populations was 54%. The mean number of alleles per polymorphic locus was 3.24 across all populations and averaged 2.37 within populations. Genetic diversity was 0.206 for the species, whereas mean population genetic diversity was 0.157, both much higher than the average for other short-lived herbaceous perennials. Estimated levels of gene flow were moderate (Nm = 0.95), and a significant association between geographic distance and genetic distance between populations was found (r = 0.68; P < 0.0001). Habitat destruction is the major threat to this genetically diverse species. Since gene flow among its highly dispersed populations is limited, diminution or extinction of local populations could jeopardize the long-term evolutionary potential of this species.     

Genetic diversity and population structure of<Emphasis Type="Italic">Kalopanax pictus</Emphasis> (Araliaceae)

Kalopanax pictus (Araliaceae) is a long-lived woody species primarily distributed throughout East Asia. This species is regarded as medically and ecologically important in Korea. We used starch gel electrophoresis to investigate its allozyme variation and genetic structure in samples from Korean populations (both natural and cultivated). Our analysis of 10 enzymes revealed 18 loci, of which 10 were polymorphic (35.6%). Genetic diversity at the species and population levels were 0.200 and 0.149, respectively, with the mean for cultivated populations (0.124) being lower than for natural (wild) populations (0.181). Asexual and sexual reproduction modes, perennial habitat, and longevity all were possible factors contributing to such high diversity. An indirect estimate of the number of migrants per generation (N _m = 1.08) indicated that gene flow was not extensive among these Korean populations. Therefore, we suggest that geographical distance as well as reproductive isolation between wild and cultivated plants may play roles in shaping the population structure of this species.     

Genetic diversity and ecological differentiation in the endangered fen orchid (<Emphasis Type="Italic">Liparis loeselii</Emphasis>)

Liparis loeselii is a rare and endangered orchid occurring in Europe and north-east America. Genetic diversity and structure of this species in north-west France and the United Kingdom were investigated using amplified fragment length polymorphisms (AFLPs). Although clonality and autogamy are common in L. loeselii, we found moderate to important variability within populations. We observed a significant genetic differentiation between populations occurring in dune slacks and fens. This may be correlated with leaf shape as dune slack individuals are sometimes treated as the distinct variety L. loeselii var. ovata. Genetic differentiation between populations was generally low suggesting that gene flow can occur over long distances and possibly across the English Channel. These results show that populations from dune slacks and fens should be managed separately and that geographically distant populations may be equivalent.     

Genetic variation and phylogeographic history of <Emphasis Type="Italic">Picea likiangensis</Emphasis> revealed by RAPD markers

Repeated cycles of retreat and recolonization during the Quaternary ice ages are thought to have greatly influenced current species distributions and their genetic diversity. It remains unclear how this climatic oscillation has affected the distribution of genetic diversity between populations of wind-pollinated conifers in the Qinghai-Tibetan region. In this study, we investigated the within-species genetic diversity and phylogenetic relationships of Picea likiangensis, a dominant forest species in this region using polymorphic DNA (RAPD) markers. Our results suggest that this species has high overall genetic diversity, with 85.42% of loci being polymorphic and an average expected heterozygosity (H _E) of 0.239. However, there were relatively low levels of polymorphism at population levels and the differences between populations were not significant, with percentages of polymorphic bands (PPB) ranging from 46.88 to 69.76%, Nei’s gene diversity (H _E) from 0.179 to 0.289 and Shannon’s indices (Hpop) from 0.267 to 0.421. In accordance with our proposed hypothesis, a high level of genetic differentiation among populations was detected based on Nei’s genetic diversity (G _ST = 0.256) and AMOVA analysis (Phi _st = 0.236). Gene flow between populations was found to be limited (Nm = 1.4532) and far lower than reported for other conifer species with wide distribution ranges from other regions. No clusters corresponding to three morphological varieties found in the south, north and west, respectively, were detected in either UPGMA or PCO analyses. Our results suggest that this species may have had different refugia during the glacial stages in the southern region and that the northern variety may have multiple origins from these different refugia.     

Twelve polymorphic microsatellite loci for <Emphasis Type="Italic">Achnatherum inebrians</Emphasis> (Poaceae)

Microsatellites are often highly variable and abundant in most complex genomes, therefore are widely used in population genetic studies. In this study, twelve polymorphic microsatellite loci were isolated and characterized for the Achnatherum inebrians, a plant abundant in grasslands of Northwest China. Characterization of 24 A. inebrians individuals form four geographically distant populations (Gansu, Qinghai, Xinjiang and Inner Mongolia provinces) showed moderate to high allelic diversity ranging from 3 to 13 alleles per locus, and the expected heterozygosity ranging from 0.41 to 0.67. No evidence of linkage disequilibrium was found for any locus pairwise comparisons. The markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow of this species. Na Chen and Yan-Zhuo Yang contributed equally to this work.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

珍稀濒危植物长叶红砂种群遗传多样性的ISSR分析

采用ISSR分子标记技术,对濒危小灌木长叶红砂(Reaumuria trigyna) 集中分布的5个种群的遗传多样性水平和遗传结构进行了研究。14条引物共检测到114个位点,其中99个为多态位点,多态位点比率为86.84％,长叶红砂种群具有较高的遗传多样性。物种水平上Shannon多样性指数(I)为0.468 8,Nei基因多样性指数(H)为0.308 4;种群水平上,多态位点比率P为77.89%,I为0.410 6,H为0.260 9,基因分化系数Gst为0.106 9,揭示了长叶红砂种群遗传变异多存在于种群内,种群间的遗传分化较小,占10.69%。 基因流(Nm)为4.178 7＞1,说明种群间的基因交流,防止了由于遗传漂变导致的遗传分化。聚类分析表明长叶红砂种群遗传距离与地理距离之间无显著的相关性。研究结果说明遗传多样性水平与物种本身特性和所处不同群落有关,濒危植物并不一定表现为遗传变异水平的降低。     

Contrasting patterns of spatial genetic structure of diploid and triploid populations of the clonal aquatic species,<Emphasis Type="Italic">Butomus umbellatus (Butomaceae)</Emphasis>, in Central Europe

Genetic diversity in a sample of an aquatic plantButomus umbellatus from 37 localities in Czechia and Slovakia was studied by analyzing six polymorphic loci in three enzymatic systems (SKDH, PGD and AAT). Diversity among ramets was low in eight populations with relatively extensive sampling (only one population possessed more than one multilocus genotype), suggesting high clonality of reproduction in these populations. However, among-population diversity was high: G = 0.782 and 0.881 for the samples of diploid and triploid populations, respectively. Heterozygosity of individual plants averaged over variable loci was also high: H = 0.554 for diploids and 0.453 for triploids. Genetic differentiation among populations was additionally studied using cluster analysis. Several populations of diploids clustered separately from all other populations, whereas another group of diploid populations clustered with some triploid populations, indicating the possibility of relatively recent, probably multiple origin of these triploid populations from their diploid progenitors. Association between matrices of Nei’s genetic distances among populations from different localities and matrices of geographic distances among these localities revealed highly significant correlation for the sample of diploid populations (r = 0.60,P < 0.001) but no significant correlation for the sample of triploid populations (r = 0.02,P = 0.593). These results indicate a spatial structure of diploid populations in accordance with the isolation by distance model, and a random distribution of genotypes among triploid populations ofB. umbellatus.     

High population differentiation and extensive clonality in a rare mallee eucalypt: Eucalyptus curtisii Conservation genetics of a rare mallee eucalypt

Microsatellite polymorphisms were analysed toassess the extent and pattern of geneticdiversity within and between isolatedpopulations of the rare mallee eucalypt, Eucalyptus curtisii. Twelve populations intotal were sampled throughout the 500 km rangeof the species in South-east Queensland.Results from analysis of 5 loci indicated ahigh degree of clonality within many of thesites, with two populations being comprised ofsingle genets. Estimates of radial growth ratessuggest that these clones may be between 4000and 9000 years old. Low overall levels ofgenetic diversity were recorded for thespecies, H _e = 0.54, however a highlysignificant amount of populationdifferentiation was observed, R _ST =0.22. There was no evidence of isolation bydistance and although there were morphologicaldifferences between some populations, this wasunrelated to molecular variation.The low level of genetic diversity and highproportion of interpopulation variation agreeswith the findings of other studies onregionally distributed eucalypts. The findingsindicate that previous estimates of theeffective population size of the species arelargely overestimated and that the individualpopulations are sufficiently differentiatedthat they should be treated as separatemanagement units. Further study is recommendedto elucidate the full extent of clonality inthe species and to conduct germination trialson seed collected from clonal populations.     

Genetic variation in island populations of tuatara (<Emphasis Type="Italic">Sphenodon</Emphasis> spp) inferred from microsatellite markers

Tuatara (Sphenodon spp) populations are restricted to 35 offshore islands in the Hauraki Gulf, Bay of Plenty and Cook Strait of New Zealand. Low levels of genetic variation have previously been revealed by allozyme and mtDNA analyses. In this new study, we show that six polymorphic microsatellite loci display high levels of genetic variation in 14 populations across the geographic range of tuatara. These populations are characterised by disjunct allele frequency spectra with high numbers of private alleles. High F _ST (0.26) values indicate marked population structure and assignment tests allocate 96% of all individuals to their source populations. These genetic data confirm that islands support genetically distinct populations. Principal component analysis and allelic sequence data supplied information about genetic relationships between populations. Low numbers of rare alleles and low allelic richness identified populations with reduced genetic diversity. Little Barrier Island has very low numbers of old tuatara which have retained some relictual diversity. North Brother Island’s tuatara population is inbred with fixed alleles at 5 of the 6 loci.     

Genetic variation and population structure in Korean endemic species: III.Hosta minor (Liliaceae)

Hosta minor, an insect-pollinated and rhizomatous herbaceous perennial, occurs in eastern and southern Korean Peninsula. AlthoughH. minor is a Korean endemic species and most populations of the species are scattered, the species maintains considerably high levels of genetic variation within the species, with a moderate level of variation (14%) found among populations. Nineteen of the 29 putative loci resolved (66%) were polymorphic within the species, the mean number of alleles per locus was 2.10 across all populations and averaged 1.68 within populations. In addition, genetic diversity was a considerably higher (for species and population level, mean estimates of genetic diversity were 0.275 and 0.230, respectively) than the average for other long-lived herbaceous perennials. Indirect estimate of the number of migrants per generation (Nm=1.03, calculated from mean G_ST) was moderate. Factors contributing to the high levels of genetic diversity found within populations ofH. minor include population maintenance via sexual and asexual modes of reproduction, primarily outcrossing breeding system, long generation time, probable ancient polyploid origin of the species, and moderate levels of seed dispersal by wind. Human disturbance in South Korea such as road and apartment constructions appears to be the major threat to this genetically diverse species.     

High genotypic diversity and strong spatial structure in populations of Trifolium alpestre with low seed production

Studies on clonal plants indicate that the proportion between clonal and sexual reproduction can be variable, depending on local habitat conditions and the biological characteristics of the species. In the present study, we assessed this question in Trifolium alpestre by assaying genetic diversity and spatial genotypic structure of natural populations with the use of allozyme markers. Populations revealed high genetic diversity as well as strong spatial structure of multilocus genotypes. The values of genetic diversity were moderately high. Spatially aggregated, identical genotypes spread up to 15 m along linear transects and across 4‐m² plots indicate extensive clonal propagation within populations. However, the existence of numerous unique and small‐sized clones reflects significant contribution from sexual reproduction. Spatially and temporarily stochastic soil disturbances have evidently opened new opportunities for the successful sexual recruitment from the permanent soil seed bank and thus counteracted losses of genetic and genotypic diversity. Seed production in all populations during the three study years was low, in average up to 1.5–2.4 seeds per shoot. The almost total lack of seed set for 57 bagged flower heads on genotypes grown in a common garden indicates that T. alpestre needs pollinators for seed production.