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 diversity and population structure of Opisthopappus longilobus and Opisthopappus taihangensis (Asteraceae) in China determined using sequence related amplified polymorphism markers

The genetic diversity and structure of four Opisthopappus longilobus and nine Opisthopappus taihangensis populations from Mts. Taihang were analyzed using SRAP markers. The results showed that O. longilobus (PPB = 95.16%, H = 0.349, I = 0.517) and O. taihangensis populations (PPB = 94.58%, H = 0.332, I = 0.504) had high genetic diversity. The genetic diversity within populations varied according to two species. Low genetic differentiation was detected among O. longilobus (Φ_ST = 0.105) and O. taihangensis (Φ_ST = 0.188) populations, respectively. Significantly genetic variation was found between O. longilobus and O. taihangensis (Φ_ST = 0.232). The dendrogram generated from pairwise genetic distance among all populations showed two distinct clusters, each corresponding to one species. A Mantel test revealed insignificant positive correlation between genetic distances and geographic distances (r_{O. longilobus} = 0.3021; r_{O. taihangensis} = 0.3658; P > 0.05). Those results indicated that self-compatibility, effective gene flow, biological traits, and historical factors may be the main factors causing geographical differentiation in the structure of O. longilobus and O. taihangensis 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.     

Fragmented habitat drives significant genetic divergence in the Chinese endemic plant,Urophysa henryi (Ranuculaceae)

Urophysa henryi (Oliv.) Ulbr., endemic to China with small populations, is known as a medicinal plant. In this study, ISSR markers were used to assess the genetic diversity and population structure throughout its entire distribution areas. Twelve primers revealed high genetic diversity at the species level (PPB = 95.6%; H = 0.3441; I = 0.5111), as well as high level of genetic differentiation (F_ST = 0.659, p < 0.001; G_ST = 0.677) and restricted gene flow (Nm = 0.239) among populations. According to the UPGMA and PCoA analysis, the 9 populations were clustered into three main groups, which were roughly in accordance with their geographical regions. In addition, a significant correlation between the genetic difference and geographic distances among populations was detected from the IBD analysis (r = 0.516, p = 0.003). These results indicated that the habitat heterogeneity and physical barriers play important roles in the modern distribution pattern and population divergence of U. henryi. However, human activities have posed serious threat to its living environment and continued survival. It is necessary to adopt some measures to restrict anthropogenic disturbances and preserve the existing populations.     

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.     

Polyploid origin,genetic diversity and population structure in the tetraploid sea lavender <Emphasis Type="Italic">Limonium narbonense</Emphasis> Miller (Plumbaginaceae) from eastern Spain

Limonium narbonense Miller is a fertile tetraploid species with a sporophytic self-incompatibility system. This sea lavender is found in coastal salt marshes which have been under intense human pressure during the past decades resulting in significant habitat fragmentation. Eleven microsatellite loci specifically designed for this species were amplified in 135 individuals from five populations. These markers were used to investigate the polyploid nature, the levels of genetic diversity and population structure in this species. L. narbonense showed high levels of genetic diversity (A = 7.82, P = 100% H _T = 0.446), consistent with its likely autotetraploid origin revealed in this study and obligate outcrossing breeding system. Inbreeding (F _IS) values were low in the three southern populations (mean F _IS = 0.062), and higher in the northern populations (mean F _IS = 0.184). Bayesian analysis of population structure revealed that populations could be grouped into two genetic clusters, one including three southern populations and the other the two northernmost ones. Individuals from the two northernmost populations showed higher admixture of the two genetic clusters than individuals from the three southern ones. A thorough analysis of microsatellite electrophoretic patterns suggests an autotetraploid origin for L. narbonense. The genetic structure revealed in this study is attributed to a recent migration from the southern area. This result suggests a net gene flow from the south to the north, likely facilitated by migratory movements of birds visiting the temporary flooded ponds occupied by L. narbonense.     

Genetic population structure of miiuy croaker (Miichthys miiuy) in the Yellow and East China Seas base on mitochondrial COI sequences

The miiuy croaker (Miichthys miiuy) is an economically important fish species in China. However, information about population structure in this species is limited. In this study, the genetic diversity and population structure of four populations of Miichthys miiuy were investigated using a mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) segment of 599 bp. A total of 35 polymorphic sites, and 31 haplotypes were defined in 109 individuals. High levels of haplotype diversity (0.731 ± 0.047) and low nucleotide diversity (0.00278 ± 0.0019) were observed in miiuy croaker populations. The results of AMOVA and pairwise fixation index (F_ST) analysis did not indicate any significant genetic differentiation among populations obtained from four sites (P > 0.05). In addition, no significant genealogical branches or clusters were recognized in the NJ tree. Analysis of neutral evolution and mismatch distribution suggested that the miiuy croaker might have experienced a recent population expansion.     

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.     

High genetic diversity but limited gene flow among remnant and fragmented natural populations of Liriodendron chinense Sarg

As a relic species, Liriodendron chinense is now recognized as an endangered species. To better understand the genetic structure and differentiation among remnant populations of L. chinense, we determined the genotypes of 14 simple sequence repeats (SSRs) loci across 318 individuals from 12 natural populations and 750 seedlings from five offspring populations. We found that L. chinense maintained high genetic diversity (H_e = 0.7385) within populations but moderate genetic differentiation (F_st = 0.1956) and low gene flow (N_m = 1.0283) between populations. The genetic diversity was slightly lower for offspring populations than for their corresponding natural populations. Moreover, using a two-phased model of mutation (TPM), we demonstrated that significant bottlenecks had occurred in six populations. A Mantel test revealed a statistically significant correlation between the geographic distances and genetic distances between populations (r = 0.5011, P = 0.002). Hence, we presume that geographical isolation and habitat fragmentation might contribute jointly to current population structure of L. chinense. We suggest that populations from southern Yunnan can be regarded as a variety of L. chinense, given their large deviation from other populations. Our findings may be of value for the conservation and use of L. chinense.     

Genetic variation within the sand-fixation species Caragana microphylla (Leguminosae) in Horqin sandy land detected by inter-simple sequence repeats analysis

Caragana microphylla is the most dominant and constructive shrub species in the Horqin sandy land in the northeast of China. We evaluated it's the level of genetic variation within and among populations sampled from two different populations types in Horqin sandy land by using inter-simple sequence repeat polymorphism (ISSR) molecular markers. The results showed that eight ISSR primers generated 106 bands, of which 87 were polymorphic. At the species level, genetic diversity was relatively high (P = 82.08%, h = 0.2831, I = 0.4233). Genetic variation in natural populations (h = 0.2152, I = 0.3169) was more than that in plantation populations (h = 0.2021, I = 0.3040). Based on Nei's G_ST value, more genetic differentiation among plantation populations was detected (G_ST = 0.7787). Six populations of C. microphylla clustered into two clades. These results have important implications for restoring and managing the degraded ecosystem in arid and semi-arid areas.     

Genetic diversity and population structure analysis of Qinghai-Tibetan plateau schizothoracine fish (Gymnocypris dobula) based on mtDNA D-loop sequences

Gymnocypris dobula is a commercially important fishery species and mostly distributed in the freshwater of Tibetan plateau. In this study, genetic diversity and intraspecific population differentiation were examined by using mitochondrial DNA D-loop sequences in 97 individuals sampled from three localities (Pali, Lasa and Yanghu) in the Tibetan plateau. Two hundred and fourteen polymorphic sites and 50 haplotypes were defined among the three localities. Genetic diversity analysis showed that the highest genetic diversity level was found in Pali population. Phylogenetic relationships analysis results indicated that closer phylogenetic relationships were found between the Yanghu and Lasa populations. Genetic population differentiation analysis indicated that the majority of variation (84.91%) was attributed to variations among populations and the largest differentiation was found between Pali and Lasa localities (F_st = 0.874, Nm = 0.036). Moreover, the historical demographic events were assessed by implementing the mismatch distribution analysis, Fajima's D test and Fu's Fs test. The results indicated that the Pali population had undergone a demographic expansion, possibly within the last 0.163 MYA (Million Years Ago). Our study firstly identified the population genetic structure of the G. dobula, which could be helpful for artificial breeding, fishery stock identification and resource conservation for this species.     

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.     

Chloroplast microsatellite diversity of Clintonia udensis (Liliaceae) populations in East Asia

Nineteen populations of Clintonia udensis Trautv. & Mey. were examined to quantify genetic diversity and genetic structure by chloroplast DNA microsatellites (cpSSR). Significant cpSSR genetic diversity (PPB = 63.64%) was detected in this species. Tetraploid populations demonstrated approximately the same level of genetic diversity as the diploid ones. A significant differentiation, however, was found between tetraploids and diploids. Most of the sixteen chloroplast haplotypes were limited to a single population. The level of haplotype diversity was high (Hd = 0.915). AMOVA, PCA and Bayesian clustering analysis revealed that there were significant genetic differences among populations. Inter-population genetic distances among population sites correlated significantly with geographic distances. These results indicate that the mixed-mating – breeding system, limited gene flow, environmental stress, and historical factors may be the main factors causing geographical differentiation in the genetic structure of C. udensis.     

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.     

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 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 of the traditional Chinese medicinal plant Ypsilandra thibetica (Melanthiaceae): Applications for conservation

Twelve microsatellite markers were developed to determine the genetic diversity and genetic structure of Ypsilandra thibetica, represented by a total of 90 individuals from six natural populations. All twelve microsatellite loci were polymorphic, and the results indicated that a high genetic diversity was present within populations (mean R_S = 4.996; mean H_E = 0.615), with high levels of genetic structure (mean F_ST = 0.165; mean F_IS = 0.692) among populations. This pattern is likely attributable to consanguineous mating, and this hypothesis is supported by a low relatedness coefficient. Our study suggested that environment factors might restrict gene flow among populations. In addition, physical distances between populations were not related to genetic distances, implying that ancestral populations might have been distributed over a wider area. These results suggest that Y. thibetica should be a high priority for conservation managers.     

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.     

Sequence-related amplified polymorphism (SRAP) of wild emmer wheat (Triticum dicoccoides) in Israel and its ecological association

Genetic diversity and population structure of 15 wild emmer wheat (Triticum dicoccoides) populations from Israel were detected by 30 sequence-related amplified polymorphism (SRAP) primer pairs. Two hundred and forty four fragments out of 438 were polymorphic. The proportion of polymorphic loci (P), the genetic diversity (He), and Shannon's information index were 0.557, 0.198, and 0.295, respectively. The population Amirim had the highest genetic variation, whereas the population of Tabigha had the lowest genetic variation. The hierarchical analysis of molecular variance (AMOVA) revealed that most of the variation was presented within populations. The value of genetic distance (D) between the populations varied from 0.027 to 0.165 with an average of 0.079, and the estimates of genetic distance were geographically independent based on the Mantel test (r = 0.105, P = 0.168). A total of 30 significant (P < 0.05) correlations were detected between 14 SRAP loci and 12 ecogeographic factors.     

Assessing the genetic consequences of flower-harvesting in Rhododendron decorum Franchet (Ericaceae) using microsatellite markers

Rhododendron decorum is widely distributed shrub in southwest China, and its flower is a favorite food of the local people. To investigate the impacts of harvesting, we genotyped 8 nuclear microsatellite loci in a total of 247 individuals from 10 natural populations and 4 flower-harvesting populations. No significant differences in allelic richness, effective number of alleles, private allelic richness, heterozygosity and effective population size were found among the natural and flower-harvesting populations. Differentiation between the 14 populations is relatively low (F_ST = 0.107). R. decorum showed high levels of intra-population genetic diversity. AMOVA analysis indicated that over 89% of the variation was contained within the populations, and that only 0.47% of the variation was attributed by human harvesting practices. Cluster analysis revealed two basic clusters related to the plants' geographical locations. Our results indicate that historical flower-harvesting practices do not lead to loss of genetic variation in R. decorum.