Genetic structure of endangered Emmenopterys henryi Oliv. based on ISSR polymorphism and implications for its conservation

Inter-simple sequence repeat (ISSR) markers were used to determine the genetic variation and genetic differentiation of nine populations of Emmenopterys henryi Oliv., an endangered plant endemic to China. Relatively low genetic diversity was detected at population level (the percentage of polymorphic loci P=22.56%, the number of alleles per locus A=1.183+/-0.045, the effective number of alleles per locus A(E)=1.007+/-0.345, Nei's gene diversity h=0.071+/-0.017, Shannon information index I=0.104+/-0.025). However, the genetic diversity at species level was relatively high (P=56.05%; A=1.561+/-0.498, A(E)=1.325+/-0.371, h=0.191+/-0.199, I=0.287+/-0.284). Analysis of molecular variance showed that most of the ISSR variation (68.03%) in E. henryi occurred among populations. The estimated Nm from F (ST )was 0.235. It indicated that the fragmentation and isolation of populations might result from specific evolutionary history and anthropogenic activity. Consequently, genetic drift might play an important role in determining the genetic structure of E. henryi. Conservation strategies for this endangered species are proposed based on the genetic data.     

Conservation genetics of an endemic and endangered epiphytic Laelia speciosa (Orchidaceae)

We used isozymes (16 loci in 11 enzymatic systems) from Laelia speciosa, an endemic and endangered epiphytic orchid of Mexico, to assess the genetic diversity and population genetic structure in nine populations distributed along its geographic range, as well as to detect those populations that are genetically unique and therefore deserve high-priority protection. On average, the genetic diversity was high (percentage of polymorphic loci, P(p) = 76%, mean number of alleles per locus, A = 3.34, the average observed heterozygosity H(O) = 0.302, the average expected heterozygosity H(E) = 0.382). Moderate levels of inbreeding (?f = 0.216, 95% confidence interval = 0.029-0.381) were found. Low levels of genetic differentiation were observed among populations ((p) = 0.040); however, there was a significant correlation between geographic and genetic distances among the populations (Mantel test: r(2) = 0.43, P < 0.05). Populations located within the same mountain range were genetically more similar. Private alleles were found, so proper management requires protection and maintenance of genetic diversity throughout its range. In case of reintroduction, we suggest using individuals propagated from seeds from as many capsules as possible, from close populations. An ex situ conservation strategy also is proposed.     

Population genetics and the conservation status of the threatened Iberian steppe grass Puccinellia pungens (Pau) Paunero (Poaceae)

Puccinellia pungens (Pau) Paunero is a narrowly endemic grass found in two continental saline lagoons of north-eastern Spain. This rare plant has been classified as 'at risk of extinction' in several national and European catalogues of endangered species. Recent demographic studies indicate that population sizes greatly exceed several million individuals, challenging that threat category. Our genetic analysis, based on allozymes, has shown that in spite of the large population sizes, very low levels of genetic variation were found in P. pungens . Genetic variation was similar in most populations, but the largest, Gallocanta lagoon as a whole, had less variation (35% polymorphic loci, 1.4 alleles/locus, H _T = 0.038) than the more restricted Royuela range (45% polymorphic loci, 1.5 alleles/locus, H _T = 0.056), suggesting a recent population expansion of the Gallocanta populations from few founder lines. The low genetic distances among populations also suggest a recent divergence. The low genetic variation observed cannot be explained fully by eventual clonal spread and rare seedling establishment in the hypersaline environment. This low variation seems to result from extreme recent population bottlenecks as a consequence of habitat conversion to agricultural fields. In the light of our data, it seems unlikely that reinforcement of populations could increase the genetic diversity of the populations. Hence, conservation efforts should focus on avoiding further habitat loss of this endangered steppe grass species.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 269–281.     

Levels and patterns of genetic variation in the endangered species Abronia macrocarpa

Genetic variation was evaluated in the federally endangered species Abronia macrocarpa (large-fruited sand-verbena), an herbaceous perennial restricted to deep sandy soils and endemic to three counties of east-central Texas. Seven of the ten known populations were sampled and analyzed using starch gel electrophoresis of eight enzymes coded by 18 interpretable loci. Duplicate gene expression was observed for four loci, suggesting polyploid ancestry for the lineage that includes A. macrocarpa. Values for estimators of genetic polymorphism within populations (ranges: P = 38.9%-61.1%, A = 1.7-2.1, H = 0.122-0.279) exceeded average values for seed plants (P = 34.2%, A = 1.53, H = 0.113). Genotype proportions at most loci in most populations were in Hardy-Weinberg equilibrium, consistent with obligate outcrossing previously documented for this species; exceptions could be attributed to population substructure. Values of F(ST) tended to be high, ranging from 0.021 to 0.481 for individual loci (mean F(ST) = 0.272), indicating substantial divergence and limited gene flow among populations, despite their close geographic proximity. Pairwise values of Nei's genetic identity between populations ranged from 0.799 to 0.975 and tended to be influenced by geographic proximity of population pairs. Collectively, these data suggest a long history of isolation among populations that have not been subjected to bottlenecks. Isolation of A. macrocarpa populations apparently results from the disjunct occurrence of suitable habitat and perhaps has been accentuated by human disturbance.     

姜状三七遗传多样性和遗传分化研究

利用5个DNA片段及12个微卫星标记（SSR）对姜状三七（Panax zingiberensis C.Y.Wu et K.W.Feng）的遗传多样性和遗传分化进行分析。结果显示，与其他人参属（Panax）植物相比：姜状三七具有相对较高物种水平的核苷酸多态性和等位基因数；在居群水平上，景谷居群具有最高的核苷酸多态性和等位基因数，而江城居群最低。AMOVA分析结果表明，姜状三七在物种水平上具有一定程度的遗传分化，但不显著；在居群水平，江城居群与其他居群间的分化程度最高，其他居群间遗传分化不明显。Structure分析结果也显示江城居群与其他居群被聚类到不同分组，且形态特征相近的样本并未聚到一起，同一分布点的样本遗传成分更相似。     

Understanding clonal diversity patterns through allozyme polymorphism in an endangered and geographically restricted Australian shrub, Zieria baeuerlenii and its implications for conservation

An electrophoretic genetic analysis utilising starch gel electrophoresis was employed to assess clonality in endangered Zieria baeuerlenii populations distributed over an area of less than a kilometre square in Nowra (NSW). Eleven enzymes systems encoded by 19 loci (41 alleles) when assayed to estimate levels of genetic diversity within and among populations, revealed moderate levels of genetic diversity. Despite finding seven loci being fixed at a particular locus, 20 unique multilocus genotypes/clones restricted to a particular population were detected within a sample of 179 ramets collected from throughout the range of the species. The probability of finding any clone produced by sexual reproduction is <0.0025 and is further supported by the fact that reproduction appears to be exclusively by vegetative spread and there is virtually no pollen viability and seed production. The lack of seed set in this area may be due to additional factors inhibiting sexual reproduction. Overall, such genetic studies play a crucial role in devising conservation and management strategies for rare and endangered taxa.     

Allozyme variation in the endangered insular endemic Castilleja grisea

BACKGROUND AND AIMS: Genetic diversity in Castilleja grisea, an endangered, perennial herb endemic to San Clemente Island, California was investigated. Subsequent to the elimination of goats from the island in 1992, many populations of C. grisea have reappeared and have been increasing in size. METHODS: Nineteen populations were surveyed for their genotype at 19 allozyme loci. KEY RESULTS: At the taxon level, 57.9 % of loci are polymorphic with A(P) = 3.09 and H(E) = 0.137. Populations averaged 33.0 % polymorphic loci with A(P) = 2.43 and H(E) = 0.099. Most variation is found within rather than among populations (G(ST) = 0.128), although differentiation among populations is significant. Genetic identities range from I = 0.960 to I = 1.000 with mean I = 0.990. There is no significant relationship between genetic and geographic distance. Gene flow among populations is Nm = 2.50 based on private alleles and Nm = 1.70 based on F(ST). Outcrossing rates based on fixation indices average t = 1.01, indicating a primarily out-crossed mating system. CONCLUSIONS: The observed genetic variation is moderately high, unusually so for an insular endemic species, suggesting that C. grisea may not have lost substantial genetic variation during 150 years of overgrazing, and indicating that it is unlikely to be endangered by genetic factors.     

Allozyme variation in endangered Castanea pumila var. pumila

Allozyme genetic variation in 12 populations of the endangered Castanea pumila var. pumila (Allegheny chinkapin), sampled across the natural range of the species in the United States, was evaluated using 11 loci from seven enzyme systems. At the species level, the percentage of polymorphic loci (Ps) was 72.7 %, the mean number of alleles per locus (As) was 1.9, the mean number of alleles per polymorphic locus (APs) was 2.3, the effective number of alleles per locus (Aes) was 1.5 and the genetic diversity (Hes) was 0.296. At the population level, Pp = 49.2 %, Ap = 1.5, Aep = 1.4, APp = 2.1 and Hep = 0.21. Most of the allozyme variation (70 %) in C. pumila var. pumila occurred within populations. Wright's gene flow rate [Nm(W)] was as low as 0.57. Population differentiation along the species range was not detected. Populations of C. pumila var. pumila in Florida had the most variable levels of genetic diversity, but populations in Virginia and Mississippi also showed high levels. Based on the results of this study, conservation management strategies are discussed.     

Allozyme variation of Cyclobalanopsis championii (Fagaceae), a narrowly distributed species in southern Taiwan

Allozyme genetic variability in five natural populations of Cyclobalanopsis championii (Fagaceae) in Taiwan was investigated using 12 loci from 9 enzyme systems. The average values of parameters describing within-population variation, expected heterozygosity (He = 0.151), the percentage of polymorphic loci per individual (P = 50%), the average number of alleles per locus (A = 1.7), effective number of alleles per locus (Ae = 1.25), and the average number of alleles per polymorphic loci (AP = 2.2) are comparable to those of other long-lived woody plants. The overall fixation index (Fis = 0.208) indicates a significant deficiency of heterozygotes at the population level. Allelic frequency deviation from Hardy-Weinberg equilibrium was found for different loci in different populations. An exact test for population differentiation using the Tools for Population Genetic Analyses program also indicates that allelic frequencies among populations are significantly different (P < .001). Among-population variation, Gst, accounted for 9.2% of the total heterozygosity. The population at Shouchia and the southernmost population Nanjenshan had higher inbreeding coefficients (0.177 and 0.153, respectively) than did the northern populations. Genetic drift is supported by the observations of the variance components of linkage disequilibrium and a large proportion of loci in Nanjenshan and Shouchia that show pairwise locus disequilibrium. We believe continuous genetic drift in the southern populations will increase genetic divergence among populations of C. championii in Taiwan. Significant correlation was found between elevation and expected heterozygosity. We therefore inferred that temperature is the most important ecological factor to influence the genetic diversity of C. championii.     

Microsatellite analysis of genetic structure in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

The level of genetic variation throughout the entire worldwide range of the mangrove species Avicennia marina (Forsk.) Vierh. was examined using microsatellite markers. Three microsatellite loci detected high levels of allelic diversity (70 alleles in total), essential for an accurate estimation of population genetic parameters. The informativeness of the microsatellite loci tended to increase with increasing average number of repeats. The levels of heterozygosity detected for each population, over all loci, ranged from 0.0 to 0.8, with an average of 0.407, indicating that some populations had little or no genetic variation, whereas others had a large amount. Populations at the extremes of the distribution range showed reduced levels of heterozygosity, and significant levels of inbreeding. This is not unexpected as these populations may be subject to founder effects and environmental constraints. The presence of genetic structure was tested in A. marina populations using three models: (i) a single panmictic model; (ii) the discrete subpopulation model; and (iii) the isolation by distance model. The discrete subpopulations model was supported by the overall measures of population differentiation based on the infinite alleles model (F-statistics), and the stepwise mutation model (R statistics). In addition, an analysis of molecular variance (AMOVA), using both theoretical models, found that most of the variation was between populations (41-71%), and within individuals in the total population (31-49%). There was little variation among individuals within populations (0-10%). There was no significant isolation by distance. The high levels of genetic differentiation observed among populations of A. marina may be due to environmental and ecological factors, particularly past sea level and climatic changes.     

Partitioning and diversity of nuclear and organelle markers in native and introduced populations of Epipactis helleborine (Orchidaceae)

Variability of allozymes (1170 individuals, 47 populations) and chloroplast DNA (692 individuals, 29 populations) was examined in native European and introduced North American populations of Epipactis helleborine (Orchidaceae). At the species level, the percentage of allozyme loci that were polymorphic (P(99)) was 67%, with a mean of 2.11 alleles (A) per locus, and an expected heterozygosity (H(exp)) of 0.294. At the population level, mean P(99) = 56%, mean A = 1.81, and mean H(exp) = 0.231. Although field observations suggest that self-pollination occurs frequently, populations had a genetic structure consistent with Hardy-Weinberg expectations and random mating (mean F(IS) = 0.002). There was significant deviation from panmixia associated with population differentiation (mean F(ST) = 0.206). The distribution of two chloroplast haplotypes showed that 15 of the 29 populations were polymorphic. Using both nuclear and organelle F(ST) estimates, a pollen to seed flow ratio of 1.43?:?1 was calculated. This is very low compared with published estimates for other plant groups, consistent with the high dispersability of orchid seeds. Finally, there was no evidence for a genetic bottleneck associated with the introduction of E. helleborine to North America.     

High levels of genetic polymorphism in the insular endemic herb Jepsonia malvifolia.

Jepsonia malvifolia is a long-lived perennial herb endemic to the Channel Islands of southern California and Guadalupe Island, Mexico. Twelve populations of J. malvifolia on San Clemente Island were surveyed for their genotype at 21 allozyme loci, revealing high levels of genetic polymorphism. For all individuals across San Clemente Island, 95.2% of loci are polymorphic with A(p) = 2.90 and H(e) = 0.179. Populations averaged 60.2% polymorphic loci with A(p) = 2.42 and H(e) = 0.158. Most variation is found within rather than among populations (G(ST) = 0.101), although differentiation among populations is significant. Genetic identities range from 0.936 to 0.999 with mean I = 0.975. There is no significant relationship between genetic and geographic distance. Gene flow among populations is Nm = 9.5 based on private alleles and Nm = 2.2 based on F(ST). Outcrossing rates based on fixation indices average t = 0.753, indicating a primarily outcrossed mating system. The genetic variation observed is unusually high for an insular endemic herb and indicates that J. malvifolia is unlikely to be endangered by genetic factors.     

Development of tetranucleotide microsatellite markers for the cushion star, Asterina gibbosa, and cross-species amplification

We describe nine polymorphic tetranucleotide microsatellite loci from the starfish, Asterina gibbosa. Loci were isolated from a partial genomic library that had been enriched for AAAC repeat sequences. Number of alleles per locus ranged from two to 14 in a sample of 85 individuals from three populations (two from Spain and one from the UK). Observed and expected heterozygosities per population ranged from 0.000 to 0.400 and from 0.040 to 0.784, respectively. All loci presented significant heterozygote deficits in one or more populations. Eight of these loci were amplified and variable in A. pancerii and A. phylactica. These loci will be used to study population structure in A. gibbosa.     

Genetic diversity in fragmented populations of Berchemiella wilsonii var. pubipetiolata (Rhamnaceae)

BACKGROUD AND AIMS: Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is distributed in fragmented habitat patches in eastern China. It is highly endangered because of severe disturbance by anthropogenic activities. Information on genetic variation and structure is critical for developing successful conservation strategies for this species. METHODS: Allozyme variation of population genetic diversity and structure was investigated for a total of 98 individuals sampled from four extant populations using isoelectric focusing in thin-layer polyacrylamide slab gels. KEY RESULTS: Based on 20 loci scored from the nine enzymes examined, a high genetic diversity was detected at both the species and population level, while there was a loss of low frequency alleles (<0.1) in all populations. Most loci showed deviation from Hardy-Weinberg equilibrium due to excess of heterozygotes in all populations, suggesting that selection for heterozygotes has occurred in this species. The genetic diversity was mainly found within populations with a moderate genetic differentiation (F(ST) = 0.13), but the two geographically discontinuous population groups showed significant differences, with F-statistic values of 0.078 for the Zhejiang populations and 0.014 for the Anhui populations, respectively. CONCLUSIONS: It appears most likely that this species has experienced a recent decrease in population size, and genetic drift in small populations has resulted in a loss of alleles occurring at low frequency. The differentiation into two population groups reflects a population genetic consequence that has been influenced by the different land-use in the two regions. Some conservation concerns are discussed together with possible strategies for implementing in situ and ex situ conservation.     

Genetic structure and conservation of Mountain Lions in the South-Brazilian Atlantic Rain Forest

The Brazilian Atlantic Rain Forest, one of the most endangered ecosystems worldwide, is also among the most important hotspots as regards biodiversity. Through intensive logging, the initial area has been reduced to around 12% of its original size. In this study we investigated the genetic variability and structure of the mountain lion, Puma concolor. Using 18 microsatellite loci we analyzed evidence of allele dropout, null alleles and stuttering, calculated the number of allele/locus, PIC, observed and expected heterozygosity, linkage disequilibrium, Hardy-Weinberg equilibrium, F(IS), effective population size and genetic structure (MICROCHECKER, CERVUS, GENEPOP, FSTAT, ARLEQUIN, ONESAMP, LDNe, PCAGEN, GENECLASS software), we also determine whether there was evidence of a bottleneck (HYBRIDLAB, BOTTLENECK software) that might influence the future viability of the population in south Brazil. 106 alleles were identified, with the number of alleles/locus ranging from 2 to 11. Mean observed heterozygosity, mean number of alleles and polymorphism information content were 0.609, 5.89, and 0.6255, respectively. This population presented evidence of a recent bottleneck and loss of genetic variation. Persistent regional poaching constitutes an increasing in the extinction risk.     

Spatial and demographic population genetic structure in Catasetum viridiflavum across a human-disturbed habitat

Spatial and temporal genetic structures were examined across sites on islands and mainland (continuous forest) populations of an epiphytic orchid, Catasetum viridiflavum, using 17 polymorphic allozyme loci. I tested whether patches on islands or at mainland sites comprised small local populations or a large population. Low among population differentiation was observed across the landscape suggesting that the species-specific pollinator and tiny wind-dispersed seeds maintain interconnections among distant patches. Temporal genetic structure among stage classes, and among breeding individuals are important components of the maintenance of genetic variation in this orchid. The natural history of this species including small breeding populations, probable high frequency of mating among relatives, and the high rates of seed movement among sites contribute to the high FIS. These data show that physically isolated patches in this epiphytic orchid comprise a single larger genetic population, which is independent of the physical distances among sites. Although quite different in ecological and life history characteristics, the genetic structure of this orchid demonstrates a pattern similar to temperate and tropical trees in fragmented landscapes.     

Population genetic structure of wild Phaseolus lunatus (Fabaceae), with special reference to population sizes

To set up an in situ conservation strategy for Phaseolus lunatus, we analyzed the genetic structure of 29 populations in the Central Valley of Costa Rica. Using 22 enzyme loci, we quantified the proportion of polymorphic loci (P(p)), the mean number of alleles per locus (A), and the mean effective number of alleles per locus (A(e)), which equaled to 10.32%, 1.10, and 1.05, respectively. The total heterozygosity (H(T)), the intrapopulation genetic diversity (H(S)), and the interpopulation genetic diversity (D(ST)) were 0.193, 0.082, and 0.111, respectively. The genotypic composition of the analyzed populations showed a deviation from the Hardy-Weinberg proportions (F(IT) = 0.932). This disequilibrium was due to either genetic differentiation between populations (F(ST) = 0.497) or nonrandom mating within populations (F(IS) = 0.866). From the level of genetic differentiation between populations and the private alleles frequencies estimates, gene flow was calculated: Nm(W) = 0.398 and Nm(S) = 0.023, respectively. The results suggested that wild Lima bean maintains most of its isozyme variation among populations. Significant positive correlation was observed between population size and P(p), A, and H(o) (observed heterozygosity), whereas no correlation was observed with the average fixation index of population (F). The loss of genetic variability in populations was attributed to inbreeding and the bottleneck effects that characterized the target populations. In situ conservation and management procedures for wild Lima bean are discussed.     

Genetic variation in remnant populations of Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

BACKGROUND AND AIMS: Dalbergia nigra, known as Brazilian rosewood, is an endangered tree species restricted to the Brazilian Atlantic Forest and has been intensively logged for five centuries due to its high-quality wood. The objective of the present study was to assess the genetic variation and structure in adults and saplings of the species from a large reserve of the Atlantic Forest, the Rio Doce State Park, and from two small surrounding fragments, one better preserved and another with a high degree of anthropogenic disturbance. METHODS: Analyses of genetic variation and structure were conducted by studying allozyme markers. Seven putative enzymatic loci were resolved, five of them being polymorphic. KEY RESULTS: The mean numbers of alleles per locus (A) were 1.93 and 1.73, while the percentages of polymorphic loci (P) were 93 and 73 % for adults and saplings, respectively. Saplings from the fragment with high anthropogenic disturbance exhibited the lowest values of A and P. The fragment that constitutes a conservation area exhibited genetic variation similar to the population from the large reserve. The observed (H(o)) and expected (H(e)) heterozygosities were not significantly different among the three populations. Only sapling populations showed F(ST) values (divergence among populations) significantly different from zero over all studied loci. The fragment with high anthropogenic disturbance exhibited considerable genetic divergence in relation to the above-cited populations. CONCLUSIONS: The evaluated populations displayed mean levels of genetic variation intermediate to those expected for narrow and widespread species. The results suggest that fragments with similar area and geographical distance from a large protected reserve can exhibit different levels of genetic variation, depending on the degree of anthropogenic disturbance. The considerable genetic variation in the protected fragment points to the importance of adequate conservation of small fragments for the preservation of genetic variation in D. nigra.     

Genetic variation and structure of the endangered Lady Fern Athyrium viridescentipes based on ubiquitous genotyping

To clarify the genetic status and provide effective information for the conservation of Athyrium viridescentipes, a critically endangered fern species with only 103 individuals remaining in the wild, we conducted ubiquitous genotyping to determine the genotypes of all remnant individuals of the target species. We analyzed the genetic variation of the 103 known individuals in four populations by using 13 microsatellite loci. The genotypes of single spores from a sporophytic individual were also determined in order to reveal the breeding system of this species. The level of allelic variation in A. viridescentipes was significantly lower than that of closely related Athyrium species. The genetic composition of the four populations was rather similar. Sixty-nine individuals (67%) possessed an identical pattern in the allele combinations at 13 microsatellite loci. The mean pairwise F (ST) among four populations was 0.018. The segregated pattern of alleles, determined by single-spore genotyping, revealed that allelic recombination occurs through meiosis. The results indicate that this species contains a low level of genetic variation, has low population differentiation, and maintains populations by sexual reproduction. These findings could lead to more effective conservation programs, the selection of the most appropriate individuals for ex situ conservation efforts, and separate management of extant populations.     

High genetic variability in an ecologically variable vertebrate,Bufo viridis

Allozymic variation in proteins encoded by 26 loci was analyzed electrophoretically in 517 specimens of green toads from 11 populations from Israel and one population from Vis Island in the Adriatic Sea. Genetic variation in this toad is the highest yet reported in any vertebrate. All three genetic parameters, mean number of alleles per locus (A), mean proportion of loci polymorphic per population (P), and mean number of heterozygous loci per individual (H), are very high (A = 1.65, range 1.38-2.04; P = 0.423, range 0.346-0.615; H = 0.133, range 0.108-0.159). Central and marginal mainland populations are only slightly more variable than desert isolates, but much more variable than the Vis Island population. Genetic similarity is very high between mainland populations (S = 0.951, range 0.93-0.97). Frequencies of two alleles (Icd-lc and Tfa) are correlated with an ecological gradient of increasing aridity. Regulatory enzymes appeared to contribute more to overall polymorphism than non-regulatory enzymes. The genetic variation observed suggests that selection for heterozygosity as an adaptive strategy is operating in the ecologically variable environment in which green toads live.