Patterns of Genetic Variation in Swertia przewalskii, an Endangered Endemic Species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Φ _ST comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory θ_B) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae): implications for its conservation

Omphalogramma souliei Franch. is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. souliei in NW Yunnan, China. The genetic diversity at the species level is low with P=42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenogamy predominated and autogamy played an assistant role in O. souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.     

Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers

Genetic variation within and between five populations of Oryza granulata from two regions of China was investigated using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pairwise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. Our results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Received: 29 March 2000 / Accepted: 15 May 2000     

Genetic Variation Within and Among Populations of Rhodiola alsia (Crassulaceae) Native to the Tibetan Plateau as Detected by ISSR Markers

Genetic variation of 10 Rhodiola alsia (Crassulaceae) populations from the Qinghai–Tibet Plateau of China was investigated using intersimple sequence repeat (ISSR) markers. R. alsia is an endemic species of the Qinghai–Tibet Plateau. Of the 100 primers screened, 13 were highly polymorphic. Using these primers, 140 discernible DNA fragments were generated with 112 (80%) being polymorphic, indicating pronounced genetic variation at the species level. Also there were high levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 63.4 to 88.6%. Analysis of molecular variance (AMOVA) showed that the genetic variation was mainly found among populations (70.3%) and variance within populations was 29.7%. The main factors responsible for the high level of differentiation among populations are probably the isolation from other populations and clonal propagation of this species. Occasional sexual reproduction might occur in order to maintain high levels of variation within populations. Environmental conditions could also influence population genetic structure as they occur in severe habitats. The strong genetic differentiation among populations in our study indicates that the conservation of genetic variability in R. alsia requires maintenance of as many populations as possible.     

The genetic structure of populations of the vulnerable aquatic macrophyte <Emphasis Type="Italic">Ranunculus nipponicus</Emphasis> (Ranunculaceae)

Ranunculus nipponicus (Makino) Nakai is a vulnerable aquatic macrophyte in the Kinki district, which is the southernmost distribution of this species in Japan. The genetic diversity and structure within and among eleven extant populations were assessed using the inter-simple sequence repeats (ISSR) polymerase chain reaction in association with combinations of propagation pattern (clonal and/or seeds) and genotypic geographical structure. In total, 53 bands were amplified, of which 18 (34%) were polymorphic. Analysis of the ISSR bands identified 46 genotypes among 81 individuals from one stream population and 72 distinct genotypes among 147 individuals in the Kinki district. An unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed some unity among upstream and downstream subpopulations within one stream and eleven populations. The Shannon index of genetic diversity was 0.109 for one stream population and 0.313 for total genetic diversity, suggesting relatively high genetic diversity. Analysis of molecular variance (AMOVA) revealed that 84.1% of the total genetic diversity occurred among populations and the remaining diversity (15.9%) occurred within populations. Significant genetic differentiation occurred among populations in the Kinki district. These results suggest that conservation of each population is important for maintaining genetic diversity of R. nipponicus in this district. An erratum to this article can be found at     

Genetic diversity and population structure of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bge in China revealed by ISSR and SRAP

Salvia miltiorrhiza Bge is a traditional Chinese medicinal herb used as an important drug to cure cardiovascular diseases. In this work, inter simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP) markers, were applied to assess the level and pattern of genetic diversity in five important cultivated populations of S. miltiorrhiza. Among these populations, 120 bands were amplified by 5 ISSR primers, of which all were polymorphic, and 110 polymorphic bands (90.16%) were observed in 122 bands amplified by 6 SRAP primers. A high levels of genetic diversity at the species level was detected with Hs = 0.1951, 0.1927 respectively. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (86.64 and 84.83% respectively) rather than among populations (13.36 and 15.17% respectively). Cluster analysis divided the five populations into two groups. The genetic relationships among populations have low correlation with their geographical distribution (Mantel test; r = 0.4870 and 0.5740 respectively). The study indicated that both ISSR and SRAP markers were effective and reliable for assessing the degree of genetic variation of S. miltiorrhiza. Our results suggested that random collecting, preserving and planting seeds without deliberate selection might be an efficient way to conserve genetic resources of medicinal plants. Their effective use was also discussed on the further breeding.     

Genetic Diversity and Genetic Structure of an Endangered Species, Trillium tschonoskii

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H_E) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.     

Genetic diversity and geographic differentiation of disjunct Atlantic and Amazonian populations of <Emphasis Type="Italic">Psychotria ipecacuanha</Emphasis> (Rubiaceae)

Ipecac (Psychotria ipecacuanha) is a perennial, medicinal herb that grows in the understory of semi-deciduous tropical forests in the Neotropics. Ipecacs present a widely disjunct distribution, with two of its three ranges occurring in Brazil. The Amazonian populations are at least 1600 km from the nearest Atlantic populations. This work used ISSR markers to compare the genetic diversity and structure of populations from the two Brazilian ranges. Lower genetic diversity in Amazon populations (P = 60.11%, Hs = 0.18) and higher genetic diversity in Atlantic populations (P = 73.94%, Hs = 0.20) were detected. Differentiation between ranges were high (θ ^B = 0.6838, G_ST-B = 0.6665). AMOVA revealed that 65.3% of the total molecular variance can be attributed to regional differences between the two ranges. Principal coordinate analyses and cluster analyses organized ipecacs at either individual or population level into two exclusive groups that correspond each to one of the two disjunct ranges, without exception. The results do not support a scenario that postulates human-mediated, long-distance dispersal events as a plausible origin for the distribution of the Brazilian ipecacs, but indicate geographic isolation as a long-standing barrier to genetic exchange and connectivity among populations from different ranges. Conservation implications are discussed.     

Low genetic diversity and significant population structuring in the relict Amentotaxus argotaenia complex (Taxaceae) based on ISSR fingerprinting

Amentotaxus, a genus of the Taxaceae, represents an ancient lineage that has long existed in Eurasia. All Amentotaxus species experienced frequent population expansion and contraction over periodical glaciations in Tertiary and Quaternary. Among them, Amentotaxus argotaenia complex consists of three morphologically alike species, A. argotaenia, Amentotaxus yunnanensis, and Amentotaxus formosana. This complex is distributed in the subtropical region of mainland China and Taiwan where many Pleistocene refugia have been documented. In this study, genetic diversity and population structuring within and between species were investigated based on the inter-simple sequence repeats (ISSR) fingerprinting. Mean genetic diversity within populations was estimated in three ways: (1) the percentage of polymorphic loci out of all loci (P) (2) Neis unbiased expected heterozygosity (He), and (3) Shannons index of phenotypic diversity. For a total of 310 individuals of 15 populations sampled from the three species, low levels of ISSR genetic variation within populations were detected, with P=4.66–16.58%, He=0.0176–0.0645 and Hpop=0.0263–0.0939, agreeing with their seriously threatened status. AMOVA analyses revealed that the differences between species only accounted for 27.38% of the total variation, whereas differences among populations and within populations were 57.70 and 14.92%, respectively, indicating substantial isolation between the patch-like populations. A neighbor-joining tree identified a close affinity between A. yunnanensis and A. formosana. Genetic drift due to small population size, plus limited current gene flow, resulted in significant genetic structuring. Low levels of intrapopulational genetic variation and considerable interpopulational divergence were also attributable to demographic bottlenecks during and/or after the Pleistocene glaciations.     

Genetic analyses of the federally endangered Echinacea laevigata using amplified fragment length polymorphisms (AFLP)—Inferences in population genetic structure and mating system

Echinacea laevigata (Boynton and Beadle) Blake is a federally endangered flowering plant species restricted to four states in the southeastern United States. To determine the population structure and outcrossing rate across the range of the species, we conducted AFLP analysis using four primer combinations for 22 populations. The genetic diversity of this species was high based on the level of polymorphic loci (200 of 210 loci; 95.24%) and Nei’s gene diversity (ranging from 0.1398 to 0.2606; overall 0.2611). There was significant population genetic differentiation (G_ST = 0.294; Ө^II = 0.218 from the Bayesian f = 0 model). Results from the AMOVA analysis suggest that a majority of the genetic variance is attributed to variation within populations (70.26%), which is also evident from the PCoA. However, 82% of individuals were assigned back to the original population based on the results of the assignment test. An isolation by distance analysis indicated that genetic differentiation among populations was a function of geographic distance, although long-distance gene dispersal between some populations was suggested from an analysis of relatedness between populations using the neighbor-joining method. An estimate of the outcrossing rate based on genotypes of progenies from six of the 22 populations using the multilocus method from the program MLTR ranged from 0.780 to 0.912, suggesting that the species is predominantly outcrossing. These results are encouraging for conservation, signifying that populations may persist due to continued genetic exchange sustained by the outcrossing mating system of the species.     

Genetic variation within the endangered mangrove species Sonneratia paracaseolaris (Sonneratiaceae) in China detected by inter-simple sequence repeats analysis

Sonneratia paracaseolaris, is a critically endangered mangrove species in China. Using inter-simple sequence repeats (ISSR) markers, we compared the genetic variation of introduced populations with that of natural populations to check whether the genetic diversity has been conserved. At the species level, genetic diversity was relatively high (P = 81.37%, He = 0.2241, and SI = 0.3501). Genetic variation in introduced populations (P = 75.78%, He = 0.2291, and SI = 0.3500) was more than that in natural populations (P = 70.81%, He = 0.1903, and SI = 0.2980). Based on Nei's G_ST value, more genetic differentiation among natural populations was detected (G_ST = 0.3591). Our data show that the genetic diversity of S. paracaseolaris was conserved in introduced populations to some extent, however, owing to the small natural populations and the threats they encountered, more plants should be planted to enlarge and restore the populations.     

Genetic Variation and Clonal Diversity of Psammochloa villosa(Poaceae) Detected by ISSR Markers

Genetic variation and clonal diversity of seven Psammochloavillosa(Poaceae) populations from northwest China were investigatedusing inter simple sequence repeat (ISSR) markers. Of the 84primers screened, 12 produced highly reproducible ISSR bands.Using these primers, 173 discernible DNA fragments were generatedwith 122 (70.5%) being polymorphic, indicating considerablegenetic variation at the species level. In contrast, there wererelatively low levels of polymorphism at the population levelwith the percentage of polymorphic bands (PPB) ranging from6.1 to 26.8. Analysis of molecular variance (AMOVA) showed thata large proportion of genetic variation (87.46%) resided amongpopulations, while only 12.54% resided among individuals withinpopulations. Clonal diversity was also high with 98 genets beingdetected from among 157 individuals using 12 ISSR primers. Theevenness of distribution of genotypes in P. villosa populationsvaried greatly, with all of the genotypes being local ones.No significant differences in genetic or clonal diversity werefound between populations in mobile or fixed dunes. The mainfactor responsible for the high level of differentiation amongpopulations and the low level of diversity within populationsis probably the clonal nature of this species, although selfingmay also affect the population genetic structure to some extent.The efficiency of ISSRs in identifying genetic individuals wasmuch higher than that of allozymes. An approximately asymptoticcorrelation was found between the number of genets detectedand the number of polymorphic loci used, suggesting that useof a high number of polymorphic bands is critical in genet identification.Copyright 2001 Annals of Botany Company Psammochloa villosa, ISSRs, genetic variation, clonal diversity     

Genetic diversity of Astragalus nitidiflorus, a critically endangered endemic of SE Spain,and implications for its conservation

Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population structure in five populations of Astragalus nitidiflorus, a critically endangered species endemic to southeast Spain. Eight primers amplified 78 bands with 40 (51.3%) being polymorphic. Statistical results indicated a low genetic diversity at the population and species level, with percentages of polymorphic bands (PPB) ranging from 28.2 to 37.2% (an average of 31.8%), and means of gene diversity (H_E) of 0.129 and 0.171 respectively. The Shannon’s index (SI) ranged from 0.160 to 0.214 at the population level and was 0.260 at the species level. A low level of genetic differentiation among populations was detected, based on the Shannon’s information index (0.297), the coefficient of genetic differentiation between populations (G_ST = 0.2418) and AMOVA analysis (Φ_ST = 0.255). The estimated gene flow (Nm) was 0.789. The high genetic connectivity found among populations of A. nitidiflorus is an evidence of a recent habitat fragmentation. In addition, a bottleneck event in the past has been revealed, with a subsequent reduction of population size and a loss of genetic variation. Based on these results, the conservation strategy of A. nitidiflorus was proposed.     

Assessment of genetic diversity and population structure of Bergenia stracheyi (Saxifragaceae) in the Western Himalaya (India)

Genetic diversity and population structure in Bergenia stracheyi, a threatened medicinal herb in the Western Himalaya of India was analysed using directed amplification of minisatellite DNA (DAMD) and inter simple sequence repeats (ISSR) markers. A total of 41 accessions of B. stracheyi representing three populations (Khillenmarg –KLM, Jalori Pass-JLP and Rohtang-RTG) were considered in the present study. The cumulative data analysis for 26 (10 DAMD + 16 ISSR) markers revealed 87.1% polymorphism. The maximum inter-population genetic distance was found between KLM and JLP, whereas the minimum genetic distance was found between RTG and JLP populations. The analysis of molecular variance (AMOVA) revealed maximum percentage of variation among individuals within populations (75%) than among the populations (25%). Clustering pattern of the three sample populations in STRUCTURE and PCoA analyses showed high genetic variation at population level. The present study revealed that distribution patterns, high altitudinal ranges, high habitat specificity, relatively high gene flow, small and isolated population size have shaped the current population structure of B. stracheyi in the Western Himalayan region. DAMD and ISSR markers have provided significant insights into characterization of B. stracheyi populations, and facilitate selection of appropriate accessions for further utilization in conservation and bioprospecting programmes.     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae):implications for its conservation

Omphalogramma souliei Franch. Is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. Souliei in NW Yunnan, China. The genetic diversity at the species level is low with P= 42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenog-amy predominated and autogamy played an assistant role in O. Souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.     

A Study of Conservation Genetics in Cupressus chengiana,an Endangered Endemic of China,Using ISSR Markers

ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (H _e) was 0.3120, effective number of alleles (A _e) was 1.5236, and Shannon’s information index (I) was 0.4740. At the population level, PPB = 48%, A _e=1.2774, H _e=0.1631, and I=0.2452. Genetic differentiation (G _st) detected by Nei’s genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (N _m ) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r=0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity.     

Genetic diversity and structure in the rare Colorado endemic plant <Emphasis Type="Italic">Physaria bellii</Emphasis> Mulligan (Brassicaceae)

Physaria bellii (Brassicaceae) is a rare, outcrossing perennial endemic to shale and sandstone outcrops along the Front Range of northern Colorado, USA. This species is locally abundant, but ranked G2/S2—imperiled because of threats to its habitat and a small number of populations—according to NatureServe’s standardized ranking system. Leaf tissue from ten populations was analyzed with ISSR (Inter-Simple Sequence Repeat) markers to discern the amount of genetic diversity and degree of population subdivision in P. bellii. Genetic diversity was moderate (0.22) and a moderately high degree of population structure was found (F _ST calculated using two algorithms ranged from 0.17 to 0.24). An AMOVA partitioned most of the variation among individuals within populations (76%), and the remainder among populations (24%). Results from a Principal Coordinates analysis were consistent with the geographic distribution of populations. A Mantel test of the correlation between genetic and geographic distances was highly significant (P < 0.001). The pattern of variation thus appears to be distributed along a gradient, and efforts to conserve this species should involve preserving enough populations so that gene flow between populations is not interrupted.     

Analysis of the genetic structure of Rhodiola rosea (Crassulaceae) using inter-simple sequence repeat (ISSR) polymorphisms

The genetic diversity and differentiation of eleven R. rosea populations from different parts of its wide area of occurrence were studied by ISSR markers. Using eight primers, 252 DNA fragments were generated, and 243 of those DNA fragments were found to be polymorphic, indicating a high genetic variability at the species level (P = 96.4%, h = 0.176, SI = 0.291). Relatively low levels of diversity were determined at the population level (P 30.6-59.1%, h 0.088-0.165, SI 0.137-0.257). AMOVA analysis revealed that the majority of the genetic variation was within populations (65.42%), and the variance among populations was 34.58%. Cluster analysis revealed two groups of R. rosea populations; these groups likely represent distinct evolutionary lines in the species, which are different in genetic structure, evolutionary history and chorological migration routes.     

Analysis of genetic variation in grass carp (Ctenopharyngodon idellus) from native and colonized regions using ISSR markers

The grass carp (Ctenopharyngodon idella), a freshwater species native to China, have been introduced in more than 100 countries. In this paper, inter simple sequence repeat (ISSR) markers were used to evaluate genetic variation within and among grass carp populations sampled from its native (China) and colonized habitats (USA, Hungary, and Japan). Twenty-two ISSR primers were used to generate 207 bands, of which 82 (39.61 %) were polymorphic. The highest genetic diversity was observed in the Yangtze River population, whereas the lowest diversity was found in the Tone River population (Japan). Compared with colonized populations, the native populations possess approximately twice as much genetic diversity. The AMOVA analysis showed a relatively high level of genetic variation within populations. Significant genetic differences were revealed both among the native populations and between pooled native and colonized populations.     

Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (H _E = 0.294 by ISSR and H _E = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (G _ST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.