Genetic diversity of the threatened Chinese endemic plant,Sinowilsonia henryi Hemsi. (Hamamelidaceae), revealed by inter-simple sequence repeat (ISSR) markers

Sinowilsonia henryi Hemsi., the only representative of the monotypic genus Sinowilsonia Hemsi. (Hamamelidaceae), is a threatened plant endemic to China with high phylogenetical, ecological and economical values. In the present study, inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 214 individuals sampled from 14 populations. Fifteen selected primers yielded a total of 178 bright and discernible bands. The genetic diversity was low at the population level (h = 0.1025; I = 0.1506; PPL = 26.7%), but quite high at the species level (h = 0.2449; I = 0.3690; PPL = 72.5%). In line with the limited gene flow (N_m = 0.3537), the hierarchical analysis of molecular variance (AMOVA) revealed pronounced genetic differentiation among populations (Φ_ST = 0.6639). Furthermore, the Mantel test revealed a significant correlation between genetic and geographic distances among populations (r = 0.688, P = 0.001), indicating the role of geographic isolation in shaping its present population genetic structure. The present patterns of genetic diversity of S. henryi were assumed to result largely from its evolutionary history and geographic factors. Based on these findings, conservation strategies were proposed to preserve this threatened plant.     

Genetic variability of the narrow endemic Rhamnus persicifolia Moris (Rhamnaceae) and its implications for conservation

Rhamnus persicifolia Moris is an endemic small tree belonging to the Rhamnus cathartica group, growing along mountainous streams of Central-Eastern Sardinia (Italy). ISSR markers were used to detect the genetic diversity within and among six populations representative of the species distribution range. In spite of the limited distribution of this endemic taxon, fairly high levels of genetic diversity were detected. Percentage of polymorphic bands (PPB), gene diversity (H_S and H_T) and Shannon information measure (Sh) were calculated both at population (PPB = 30.70%, H_S = 0.1105, Sh = 0.1646) and at species level (PPB = 68.42%, H_T = 0.2066, Sh = 0.3139).The existence of a spatial distribution of genetic diversity in R. persicifolia was revealed by a low gene flow, a fairly high level of genetic differentiation (G_ST = 0.4583) among populations and a positive correlation between genetic and geographic distances (Mantel test, r = 0.71, p = 0.016). The spatial genetic structure was also confirmed with BAPS analysis. Our results show that a certain level of isolation by distance and sex-ratio bias may explain the distribution of genetic diversity among populations.Conservation measures are suggested on the basis of the genetic diversity detected, by implementing an integrated in situ and ex situ conservation program for each population, in order to ensure effective protection for this endemic species.     

Genetic diversity of the endangered Chinese endemic plant Monimopetalum chinense revealed by amplified fragment length polymorphism (AFLP)

Monimopetalum chinense Rehd. is an endangered woody vine endemic to eastern China. Using amplified fragment length polymorphism (AFLP) markers, we examined levels of genetic variation within and among eleven populations located across the species’ distribution. Although modest levels of heterozygosity were detected, other measures of genetic diversity registered relatively high levels of variability, both at the species level (P = 91.0%, H_E = 0.232, I_S = 0.365) and at the population level (P = 53.0%, H_E = 0.155, I_S = 0.239). Populations also exhibited high levels of genetic differentiation (Nei’s genetic diversity analysis, G_ST = 0.330), corresponding to isolation-by-distance and hierarchical population structure. These results indicate that, despite low levels of gene flow, populations of M. chinense still harbor substantial amounts of genetic diversity. Management plans for the species should include measures that ensure genetic diversity remains high within and among extant populations.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

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.     

Genetic diversity and population structure of Swertia tetraptera (Gentianaceae), an endemic species of Qinghai-Tibetan Plateau

Swertia tetraptera Maxim is an annual alpine herb endemic to the Qinghai-Tibetan Plateau (QTP). Its populations are locally scattered as isolated patches throughout this region. Genetic variation within and among thirty-four populations of this species was assessed using ISSR fingerprinting with 10 primers. High levels of genetic diversity exist within species (P = 98.9%, I = 0.3475; He = 0.2227), while the within-population diversity is low (P = 32.7%, I = 0.177; He = 0.12). High levels of genetic differentiation were detected among populations based on various statistics, including Nei’s genetic diversity analysis (G_ST = 0.4608), Bayesian analysis (θ^B = 0.476) and AMOVA (F_ST = 0.57). That is, populations shared low levels of genetic identity (I = 0.2622–0.0966). This genetic structure was probably due to severe genetic drift, breeding system and limited gene flow. The observed genetic structure of the populations implies that different populations across the distribution range of the species should be sampled to maintain high genetic diversity when a conservation strategy is implemented.     

Genetic diversity and population structure of the red stingray,Dasyatis akajei inferred by AFLP marker

To examine population genetic diversity and variation of the red stingray Dasyatis akajei, samples from 1 freshwater region and 6 coastal localities within its distribution range were analyzed by using amplified fragment length polymorphism (AFLP) technology. A total of 207 loci were identified by 4 primer combinations from 87 individuals, 174 of which were polymorphic (84.1%). A high level of Nei's gene diversity was observed with the overall value of 0.230 ± 0.179. No significant genealogical clusters associated with sampling sites were revealed on the UPGMA tree. Both analysis of molecular variance (AMOVA, Φ_ST = 0.085, P = 0.00) and pairwise F_ST indicated significant genetic differentiation among four marine samples. However, no particular genetic differentiation was detected between marine and the limited sampling freshwater populations (AMOVA, Φ_CT = 0.056, P > 0.05). Except for the TZ vs. WZ (5.193), the gene flow estimates (N_m) demonstrated the effective immigrants were 1.918-2.976, suggesting low level dispersal between pairwise marine populations. Species-specific habits (demersal and sluggish habits) are probably responsible for the population structuring.     

Genetic diversity in Mentha cervina based on morphological traits,essential oils profile and ISSRs markers

Morphological, phytochemical and genetic differences were studied to evaluate the level and distribution of diversity in twelve populations of the Portuguese endangered medicinal plant Mentha cervina L. Morphological variation was correlated with ecological conditions at the site of origin. Pulegone was the major essential oils compound in all of the populations collected at full flowering (68–83%), in different growing conditions (51–82%), and for all the developmental stages studied (47–82%). Although clusters were defined, the analysis revealed a high chemical correlation among all populations (S_corr ≥ 0.95%). Inter-simple sequence repeats markers were used to assess the population structure and genetic variation. Populations exhibited a relatively low genetic diversity (PPB = 14.3–64.6%, H_e = 0.051–0.222, I = 0.076–0.332), with high structuring between them (G_ST = 0.51). However, the genetic diversity at species level was relatively high (PPB = 97.7%; H_e = 0.320). The levels and patterns of genetic diversity were assumed to result largely from a combination of evolutionary history and its unique biological traits, such as breeding system, clonal growth, low capacity of dispersion and habitat fragmentation. The relatively low genetic diversity in the populations analyzed indicates that the maintenance of their evolutionary potential is at risk if population sizes are maintained and if there is no protection of the habitats.     

Population genetic structure of the Korean threadsail filefish (Stephanolepis cirrhifer) based on microsatellite marker analysis

The threadsail filefish Stephanolepis cirrhifer is one of the most important commercial fish resources in Korea. However, its natural populations have declined drastically over the last several decades. To investigate the genetic characteristics of the threadsail filefish for conservation and management purposes, its genetic variation was analyzed in Korean fishing grounds using 11 microsatellite loci. All populations showed moderate genetic diversity (mean number of alleles (N_A) = 8.6, expected heterozygosity (He) = 0.73), representing a slightly lower level of genetic variability than other congeneric species. The presence of a weak genetic population substructure was detected (F_ST = 0.023, R_ST = 0.030, P < 0.001), but this substructure did not feature significant isolation based on the distance between populations. This differentiation may be primarily attributable to genetic differences between populations from Geomundo and other localities, based on relative low level of gene flow, a high level of pairwise F_ST and R_ST or the position of this population in a phylogenetic tree. An analysis of molecular variance (AMOVA) also revealed a greater part of the variation within the population and genetic differentiation among the samples analyzed and identified two genetic clusters. The possibility of a recent genetic bottleneck was observed in some populations of S. cirrhifer. Given the prevailing ocean currents and the intensity of threadsail filefish harvesting activities in Korea, the possibility that human harvest and stock enhancement activities may have partially contributed to our detected genetic pattern cannot be excluded. Management strategies that take these findings into account might lower the risk of harmful genetic change in the species.     

Genetic diversity within and among populations of the endangered and endemic species Primula merrilliana in China

Primula merrilliana Schltr. is an endangered and narrowly-distributed endemic species of southern Anhui Province in China. In this study, the level of genetic variation and the pattern of genetic structure in six natural populations of P. merrilliana were assessed by using ISSR (inter-simple sequence repeats) markers. Based on ten primers, 137 clear and reproducible DNA fragments were generated, of which 109 were polymorphic. The statistical results indicated that there was a relatively low genetic diversity within populations, and a high genetic differentiation among populations (G_ST = 0.53, Φ_ST = 0.49). The level of population genetic diversity was correlated to habitat type and the gene flow (N_m) was low with only 0.45. The unexpected genetic structure of P. merrilliana may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self-compatible breeding system and biennial life form.     

Genetic diversity of a dominant species Stipa bungeana and its conservation strategy in the Loess Plateau of China

In order to investigate the genetic diversity and population structure of Stipa bungeana under the background of grassland utilization and grazing exclusion, ten S. bungeana populations were selected from different steppe type in the Loess Plateau of China. Sequence-related amplified polymorphism (SRAP) marker was used to assess the genetic diversity. Fifteen SRAP primer combinations generated a total of 482 amplification bands, 418 (86.72%) were polymorphic bands. A relatively high level of genetic diversity (PPB = 89.80%, h = 0.1972, H = 0.3154) was detected at the species level, but the genetic diversity was low at the population level (PPB = 17.01–33.33%, h = 0.0438–0.0967, H = 0.0361–0.1447). AMOVA analysis revealed a high level of genetic differentiation among populations (Φ_ST = 0.6757), and a limited among-population gene flow (N_m = 0.1200). There was no significant correlation between genetic distance and geographic distance by Mantel test (r = 0.1126, P = 0.204). Conservation implications were proposed for S. bungeana.     

Genetic diversity of the endangered Chinese endemic herb Saruma henryi Oliv. (Aristolochiaceae) and its implications for conservation

Saruma henryi Oliv., the only representative of the monotypic genus Saruma Oliv. (Aristolochiaceae), is an endangered perennial herb endemic to China. It is a phylogenetically, ecologically, and medicinally important species. In the present study, inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 14 populations. A total of 16 selected primers yielded 175 bright and discernible bands, with an average of 10.94 per primer. POPGENE analysis showed that the genetic diversity was quite low at the population level (h = 0.0447–0.1243; I = 0.0642–0.1853; PPB = 10.29–36.57%), but pretty high at the species level (h = 0.2603; I = 0.3857; PPB = 73.71%). The hierarchical analysis of molecular variance (AMOVA) revealed a high level of genetic differentiation among populations (67.18% of total variance components, P < 0.001), in line with the gene differentiation coefficient (G _ST = 0.6903) and the limited among-population gene flow (N _m = 0.2243). Both Principal Coordinates Analysis (PCoA) and UPGMA cluster analysis supported the grouping of all 14 populations into three geographic groups, among which there occurred a moderate level of genetic differentiation (33.18% of total variance components, P < 0.001) as shown by AMOVA analysis. In addition, Mantel test revealed a significant correlation between genetic and geographic distances among populations (r = 0.7792, P = 0.001), indicating the role of geographic isolation in shaping its present population genetic structure. The present levels and patterns of genetic diversity of S. henryi were assumed to result largely from its breeding system, geographic isolation, clonal growth, its unique biological traits and evolutionary history. The high genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered herb.     

Highly differentiated populations of the narrow endemic and endangered species Primula cicutariifolia in China,revealed by ISSR and SSR

The perennial herb Primula cicutariifolia Pax is an endangered and endemic species with narrow distribution in eastern Anhui and Zhejiang provinces of China. In this study, the levels of genetic variation and the pattern of genetic structure in five natural populations of P. cicutariifolia were assessed by inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers. Both markers revealed that there was remarkably low genetic variation within populations (e.g., H_e = 0.19 and 0.18, for ISSR and SSR respectively) and high differentiation among populations (G_ST = 0.714 and 0.611; Φ_ST = 0.698 and 0.599, for ISSR and SSR respectively). The level of population genetic diversity was correlated to population size only detected by ISSR markers. The genetic structure of P. cicutariifolia may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self breeding system and biennial life form. To protect and avoid disappearance of P. cicutariifolia, much more attentions should be paid to protect all the remnant populations and their habitats, and three management units, i.e. Tianmushan, Damodao, and Panan units, were proposed.     

Genetic diversity and structure of Pinus dabeshanensis revealed by expressed sequence tag-simple sequence repeat (EST-SSR) markers

Assessing patterns of genetic variation in rare endangered species is critical for developing both in situ and ex situ conservation strategies. Pinus dabeshanensis Cheng et Law is an endangered species endemic to the Dabieshan Mountains of eastern China. To obtain fundamental information of genetic diversity, population history, effective population size, and gene flow in this species, we explored patterns of genetic variation of natural populations, in addition to an ex situ conserved population, using expressed sequence tag-simple sequence repeats (EST-SSR) markers. Our results revealed moderate levels of genetic diversity (e.g., H_E = 0.458 vs. H_E = 0.423) and a low level of genetic differentiation (F_ST = 0.028) among natural and conserved populations relative to other conifers. Both contemporary and historical migration rates among populations were high. Bayesian coalescent-based analyses suggested that 3 populations underwent reductions in population size ca. 10,000 yr ago, and that two populations may have experienced recent genetic bottlenecks under the TPM. Bayesian clustering revealed that individuals from the ex situ population were largely assigned to the ‘red’ cluster. Additionally, our results identified private alleles in the natural populations but not in the ex situ population, suggesting that the ex situ conserved population insufficiently represents the genetic diversity present in the species. Past decline in population size is likely to be due to Holocene climate change. Based on the genetic information obtained for P. dabeshanensis, we propose some suggestions for the conservation and efficient management of this endangered species.     

Genetic diversity and population structure of Taxus cuspidata in the Changbai Mountains assessed by chloroplast DNA sequences and microsatellite markers

Taxus cuspidata, a tree species with high economic value because of its anticancer properties, is experiencing severe reduction in populations across its range in China. We examined one chloroplast DNA (cpDNA) region (petA-psbE) and 9 nuclear simple sequence repeats (SSRs) loci variations among seven populations in the Changbai Mountains of China to investigate the levels of genetic diversity and population structure. A moderate level of haplotype diversity (H_T = 0.625), low nuclear microsatellite diversity (H_E = 0.261 ± 0.028), significant genetic differences (F_ST = 0.065) and substantial gene flow (Nm = 2.806) were observed. Most of the total genetic variation was partitioned within the population (87.8% and 94.0% for cpDNA and SSRs, respectively). Our haplotype identification permutation tests revealed that G_ST > N_ST, indicating an absence of phylogeographic structure in T. cuspidata. Neither STRUCTURE nor UPGMA analyses showed any geographic pattern in T. cuspidata populations. By comparatively analyzing the genetic diversity and survival situation of T. cuspidata, our results provide a theoretical foundation for the resource protection, utilization cultivation and breeding of this valuable plant.     

Assessing the genetic diversity and population structure of Culter alburnus in China based on mitochondrial 16S rRNA and COI gene sequences

The genetic diversity and population genetic structure of Culter alburnus were investigated using mitochondrial cytochrome c oxidase subunit I (COI) and ribosomal 16S subunit (16S rRNA) gene sequences. A total of 89 individuals from four localities were included in the analysis. Overall, 12 polymorphic sites were observed and 10 haplotypes were defined. The C. alburnus populations were characterized by high haplotype diversity (0.587 ± 0.047) and low nucleotide diversity (0.00197 ± 0.00073). Pairwise fixation index (F_ST) analysis indicated significant genetic differentiation among different populations. The hierarchical analysis of molecular variance (AMOVA) analysis also showed significant genetic divergence (φ_ST = 0.3792, P < 0.01) among these populations. The present results suggest that subdivisions exist among four C. alburnus populations, and should be considered as different management unit for effective conservation and management purposes. This study provides new information for genetic assessment and will be crucial for establishing fisheries management and strategies for this species.     

Genetic diversity and relationships among wild and cultivated Stenocereus queretaroensis populations in western Mexico

Stenocereus queretaroensis is an endemic, chiropterophilous, columnar cactus of economic importance in Mexico. To investigate the effect of artificial selection on genetic diversity, inter-simple sequence repeat (ISSR) markers were used to estimate the genetic variation of wild, orchard, and backyard populations of S. queretaroensis from two regions in western Mexico. Six primers were used to generate 62 bands, of which 39 were polymorphic (62.9%). The total genetic diversity was similar in the wild (H_T = 0.296) and orchard (H_T = 0.291) groups, and slightly lower in the backyard group (H_T = 0.281). Wild populations (F_ST = 0.13) were less differentiated than backyard (F_ST = 0.17) and orchard (F_ST = 0.21) populations. The wild and backyard groups were genetically closer (0.015) than the wild and orchard (0.018) groups. A Mantel test revealed a positive correlation between genetic and geographic distances (r = 0.433, p = 0.002). In conclusion, gene flow and the prevailing management system have efficiently maintained genetic diversity and facilitated inter-population differentiation in S. queretaroensis.     

Low genetic diversity and significant structuring in the endangered Mentha cervina populations and its implications for conservation

Eighteen populations of the endangered aromatic and medicinal plant Mentha cervina (Lamiaceae) were sampled across its natural range, in the western half of the Iberian Peninsula, and inter-simple sequence repeats (ISSRs) markers were used to assess genetic diversity and population structure. M. cervina populations exhibited a relatively low genetic diversity (percentage of polymorphic loci PPB = 14.2–58.3%, Nei's genetic diversity H_e = 0.135–0.205, Shannon's information index I = 0.08 − 0.33). However, the genetic diversity at species level was relatively high (PPB = 98.3%; H_e = 0.325; I = 0.23). The results of the analysis of molecular variance indicated very structured populations, with 50% of the variance within populations, 44% among populations and 6% between regions defined by hydrographic basins, in line with the gene differentiation coefficient (G_ST = 0.532). A Mantel test did not find significant correlation between genetic and geographic distance matrices (r = 0.064), indicating that isolation by distance is not shaping the present genetic structure. The levels and patterns of genetic diversity in M. cervina populations were assumed to result largely from a combination of evolutionary history and its unique biological traits, such as breeding system, low capacity of dispersion, small effective size and habitat fragmentation. The high genetic differentiation among populations indicates the necessity of conserving the maximum possible number of populations. The results also provide information to select sites for ex situ conservation. Optimal harvesting strategies, cultivation and tissue culture should also be developed as soon as possible to guarantee sustainable use of the species under study.     

Genetic structure of Pyrenophora teres net and spot populations as revealed by microsatellite analysis

The population structure of the fungal pathogen Pyrenophora teres, collected mainly from different regions of the Czech and Slovak Republics, was examined using a microsatellite analyses (SSR). Among 305 P. teres f. teres (PTT) and 82 P. teres f. maculata (PTM) isolates that were collected, the overall gene diversity was similar (? = 0.12 and ? = 0.13, respectively). A high level of genetic differentiation (F_ST = 0.46; P < 0.001) indicated the existence of population structure. Nine clusters that were found using a Bayesian approach represent the genetic structure of the studied P. teres populations. Two clusters consisted of PTM populations; PTT populations formed another seven clusters. An exact test of population differentiation confirmed the results that were generated by Structure. There was no difference between naturally infected populations over time, and genetic distance did not correlate with geographical distance. The facts that all individuals had unique multilocus genotypes and that the hypothesis of random mating could not be rejected in several populations or subpopulations serve as evidence that a mixed mating system plays a role in the P. teres life cycle. Despite the fact that the genetic differentiation value between PTT and PTM (F_ST = 0.30; P < 0.001) is lower than it is between the populations within each form (F_ST = 0.40 (PTT); F_ST = 0.35 (PTM); P < 0.001) and that individuals with mixed PTT and PTM genomes were found, the two forms of P. teres form genetically separate populations. Therefore, it can be assumed that these populations have most likely undergone speciation.     

Maintenance of genetic diversity in Cordia africana Lam., a declining forest tree species in Ethiopia

We assessed genetic variation in a total of 22 populations of the tree species Cordia africana Lam. in Ethiopia and analyzed the country-wide impact of population history, forest disturbance and alteration of land use on extant intraspecific diversity. Amplified Fragment Length Polymorphisms (AFLPs) and chloroplast microsatellite markers were investigated. The analyses of the AFLP data revealed high diversity in all investigated populations: the percentage of polymorphic loci (PPL) ranged from 62.2% to 92.2% and Nei's gene diversity from 0.220 to 0.320 within the populations. The mean PPL and the mean diversity within populations were 85.7% and 0.287, respectively. The analysis of molecular variance revealed a moderate level of differentiation (Φ_ST = 0.07, p < 0.001) among the populations. The Mantel test proved a significant but low correlation (r = 0.31, p < 0.001) between the geographic distance and the genetic differentiation matrices. Only three different cpDNA haplotypes were observed; no more than two haplotypes were found in any population. The dominant haplotype with an overall frequency of 81% was observed in all populations. The level of differentiation among the populations was comparatively low at chloroplast DNA (G _ST = 0.18, R _ST, N _ST = 0.22). The observed patterns and levels of genetic variation within and among the populations indicate that efficient gene flow via pollen and seed is likely to be the main factor contributing to the maintenance of genetic diversity in natural and disturbed conditions.