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 structure in peripheral Western European populations of the endangered species Cochlearia pyrenaica (Brassicaceae)

Cochlearia pyrenaica is one of the most endangered plant species in Europe, listed in many European and regional conservation policy documents (e.g. Spain, France, Belgium, Switzerland). To study its genetic structure, define its conservation units and propose a management strategy for this species, amplified fragment length polymorphism markers were used to analyse the genetic diversity within and between five representative populations of the species distribution in Western Europe (Cantabrian Range, North of Spain; Pyrenees, France; Wallonia, Belgium). Low levels of genetic diversity were revealed by the population percentage of polymorphic bands (PPB?=?36.56%), average within-population diversity (H _S?=?0.0990) and genetic diversity within populations (H _pop?=?0.1541), although high levels were reported at species level (PPB?=?81.16%; total genetic diversity for the species, H _T?=?0.0990; and genetic diversity within whole species, H _sp?=?0.2515). The coefficient of genetic differentiation among populations (G _ST) was 0.3869. The analysis of Shannon diversity index in population and for the total data set partitioned (38.72%) and AMOVA (53%) detected a high level of interpopulation diversity, in broad agreement with the result of genetic differentiation analysis. NeighborNet network and principal coordinate analyses clustered the populations in three major groups congruent with geographical regions. Bayesian clustering also confirmed these three distinct genetic clusters. The level of gene flow (Nm) was estimated as 0.3961 individuals per generation among populations, with the genetic identity (I) and genetic distance (D) among populations ranging from 0.8679 to 0.9651 and from 0.0355 to 0.1417, respectively. Therefore, the low levels of genetic variation and high divergence of regional gene pools indicate that there is a need to protect each disjunct region of Western Europe.     

Genetic structure of Rhamnus glaucophylla Sommier endemic to Tuscany

Rhamnus glaucophylla Sommier is an endemic small tree or shrub belonging to sect. Eurhamnus. It grows on cliffs of the Apuan Alps and of the Lucca Apennine in Tuscany (Italy). ISSRs were used to detect the genetic diversity within and among five populations representative of the species distribution range. Moderate levels of genetic diversity were revealed as percentage of polymorphic band (PPB) both at the population (PPB?=?22.63%, H _S?=?0.0801, Sh?=?0.1193) and species level (PPB?=?48.68%, H _T?=?0.1155, Sh?=?0.1837). The correlation between genetic and geographic distances was not significant (Mantel test, r?=?0.17; P?<?0.26). The information on the pattern of genetic variation obtained in this work bears important implications for conservation management, and in particular for ex situ conservation programmes involving long-term seed storage in seed banks.     

Genetic Diversity and Structure of Sinopodophyllum hexandrum (Royle) Ying in the Qinling Mountains,China

Sinopodophyllum hexandrum is an important medicinal plant whose genetic diversity must be conserved because it is endangered. The Qinling Mts. are a S. hexandrum distribution area that has unique environmental features that highly affect the evolution of the species. To provide the reference data for evolutionary and conservation studies, the genetic diversity and population structure of S. hexandrum in its overall natural distribution areas in the Qinling Mts. were investigated through inter-simple sequence repeats analysis of 32 natural populations. The 11 selected primers generated a total of 135 polymorphic bands. S. hexandrum genetic diversity was low within populations (average H_e = 0.0621), but higher at the species level (H_e = 0.1434). Clear structure and high genetic differentiation among populations were detected by using the unweighted pair group method for arithmetic averages, principle coordinate analysis and Bayesian clustering. The clustering approaches supported a division of the 32 populations into three major groups, for which analysis of molecular variance confirmed significant variation (63.27%) among populations. The genetic differentiation may have been attributed to the limited gene flow (N_m = 0.3587) in the species. Isolation by distance among populations was determined by comparing genetic distance versus geographic distance by using the Mantel test. Result was insignificant (r = 0.212, P = 0.287) at 0.05, showing that their spatial pattern and geographic locations are not correlated. Given the low within-population genetic diversity, high differentiation among populations and the increasing anthropogenic pressure on the species, in situ conservation measures were recommended to preserve S. hexandrum in Qinling Mts., and other populations must be sampled to retain as much genetic diversity of the species to achieve ex situ preservation as a supplement to in situ conservation.     

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.     

Spatial Genetic Structure of Campanula sabatia, a Threatened Narrow Endemic Species of the Mediterranean Basin

Campanula sabatia is endemic to NW Italy (Liguria) and it was included in the European Red list of endangered species due to the heavy human pressure on its habitat. AFLP markers were used to detect the genetic diversity within and among ten populations (totalling 83 individuals) representing the range of the species. In spite of the limited distribution of this endemic taxon, high levels in percentage of polymorphic bands (PPB), gene diversity (H _S and H _T) and Shannon??s information index (I) were detected both at population (PPB?=?60?%, H _S?=?0.1853, I?=?0.2836) and at species level (PPB?=?100?%, H _T?=?0.2415, I?=?0.3871). The coefficient of genetic differentiation among population (G??_ST) was 0.1935, while the level of gene flow (N?? _m) was estimated to be 2.0832. AMOVA analysis identified a genetic variation within populations of 83?% of the total. Bayesian clustering methods assigned individuals to two geographical groups partly found within the same population, probably due to a high rate of genetic exchange among its populations. Conservation measures are suggested on the basis of the genetic diversity detected to ensure an effective protection for this endemic species.     

Genetic diversity of Sinojackia dolichocarpa (Styracaceae), a species endangered and endemic to China,detected by inter-simple sequence repeat (ISSR)

Sinojackia dolichocarpa, a species endangered and endemic to China, is distributed only in the regional area of Shimen and Sangzhi in Hunan Province. Inter-simple sequence repeat (ISSR) markers were used to investigate the genetic diversity within and among the four natural populations of S. dolichocarpa. Leaf samples were collected from 84 individuals. Thirteen ISSR primers selected from 80 primers gave rise to 137 discernible DNA bands of which 100 (72.99%) were polymorphic. On average each primer gave rise to 10.5 bands including 7.7 bands with polymorphic profile. At the species level, high genetic diversity was detected (PPB: 72.99%; H_E: 0.2255; Ho: 0.3453). However, relatively low genetic diversity existed within populations. Population in Maozhuhe (MZH) exhibits the greatest level of variability (PPB: 40.38%, H_E: 0.1566, Ho: 0.2330), whereas the population in Jingguanmen (JGM) finds its own variability at the lowest level (PPB: 30.66%; H_E: 0.1078; Ho: 0.1601). A high level of genetic differentiation among populations was revealed by Nei's gene diversity statistics (45.30%), Shannon's information measure (45.24%) and analysis of molecular variance (AMOVA) (52.88%). The main factors responsible for the high level of differentiation among populations are probably related to the selfing reproductive system and the isolation of populations. The strong genetic differentiation among populations indicates that the management for the conservation of genetic variability in S. dolichocarpa should aim to preserve every population.     

AFLP and ISSR analysis reveals high genetic variation and inter-population differentiation in fragmented populations of the endangered Litsea szemaois (Lauraceae) from Southwest China

Litsea szemaois (Lauraceae) is an endemic and endangered species from the tropical rain forests of Xishuangbanna, southern Yunnan, SW China, but habitat fragmentation, especially exacerbated by rubber planting, has caused a decline in population size of the species. AFLP and ISSR were used to investigate the genetic diversity and population structure of eight populations from across its known distribution. Three AFLP and ten ISSR primer combinations produced a total of 203 and 77 unambiguous and repeatable bands respectively, of which 164 (80.8%) and 67 (87.0%) were polymorphic for the two markers. These two markers showed that Litsea szemaois exhibits comparatively high genetic diversity at species level (heterozygosity (hs) = 0.2109) relative to some other Lauraceae. Most of the genetic variation was partitioned within populations, but genetic differentiation between populations was significant and relatively high (Φ _st = 0.2420, θ^B = 0.1986) compared with other tropical plants. The genetic characteristics of L. szemaois may be related to its outbreeding system, insect pollination and fragmented distribution. Because L. szemaois is dioecious and slow to mature, ex situ conservation across its genetic diversity is unlikely to succeed, although seedlings grow well under cultivation. Thus, in situ conservation is very important for this endangered species, especially as only 133 adult individuals are known in the wild. In particular, the Nabanhe and Mandian populations should be given a high conservation priority due to their higher genetic diversity, larger population size and better field condition, but wider sampling is required across all populations to determine additional areas with significant genetic conservation value.     

Conservation genetics of the endangered terrestrial orchid Liparis japonica in Northeast China based on AFLP markers

Levels of genetic diversity and population genetic structure of the rare, endangered terrestrial orchid Liparis japonica were examined for eight natural populations (n = 185) in Northeast China using six AFLP primer pairs, where this species has experienced severe habitat loss and fragmentation. Based on 406 DNA bands, a high level of genetic diversity was found at the species level with the PPB of 85.47 %, while the genetic diversity at the population level was low (PPB = 47.48 %). A significantly high degree of population differentiation was found with 42.69 % variation existed among populations as measured by AMOVA, indicating potential restricted gene flow. The genetic distances between populations were independent of the corresponding geographic distances, and the genetic relationship of individuals had no significant correlation with their spatial distribution. The restricted gene flow might be impacted by reduced population size, habitat destruction and fragmentation. The results in this study suggested that habitat protection and keeping a stable environment are critical for the conservation of L. japonica species.     

Population genetic diversity and divergence of the halobiotic herb Limonium sinense estimated by AFLP and ISSR, and implications for conservation

Limonium sinense is a halobiotic herb endemic to China that has been traditionally used for hundreds of years for its good restorative function. Genetic variation and population structure of this species were investigated by using amplified fragment length polymorphisms (AFLPs) and inter simple sequence repeats (ISSRs). A high level of genetic diversity was detected [AFLP: H _E = 0.284, percentage of polymorphic loci (PPL) = 92.68 %; ISSR: H _E = 0.257, PPL = 85.71 %] at the species level with POPGENE. Based on analysis of molecular variation (AMOVA), the among-population component accounted for 29.03 % (AFLP) and 28.81 % (ISSR) of the genetic variation, indicating that most of the genetic variation was between individuals within populations. The Shannon diversity index (I) was higher for AFLP (0.432) than for ISSR (0.395). Five main clusters were shown in the unweighted pair-group method with arithmetic mean (UPGMA) dendrogram created using TFPGA, consistent with the result of principal coordinate analysis using NTSYS. In situ conservation is advocated first. Keeping a stable environment for this halobiotic herb is necessary. For ex situ conservation, it is important to establish a germplasm bank. AFLP and ISSR markers were proved to be efficient tools in assessing the genetic variation among populations of L. sinense. The patterns of variation appeared to be consistent for these two marker systems, and they can be used for management of genetic structure, protection of the halobiotic plant, and conservation of germplasm.     

Genetic diversity within and among populations of Neopicrorhiza scrophulariiflora (Scrophulariaceae) in China,an endangered medicinal plant

Neopicrorhiza scrophulariiflora Pennel (Hong) (Scrophulariaceae) is an endangered medicinal plant, endemic to the Eastern Himalayas and the Hengduan Mountains. Levels of genetic variation and genetic structure of seven populations of N. scrophulariiflora in China were studied using inter-simple sequence repeat (ISSR) markers. Thirteen primers amplified 82 total loci from 7 populations composed of 136 individuals. All 82 loci were polymorphic, showing a percentage of polymorphic bands (PPB) of 100%, indicating considerable genetic variation at the species level. In contrast, a low level of genetic diversity within populations was detected with a mean PPB of 30.56%. In addition, Nei’s genetic diversity analysis (0.4073) and Shannon’s diversity index (0.5917) revealed similar genetic structure. High levels of genetic differentiation (Gst = 0.6955) and restricted gene flow (Nm = 0.2198) among populations were also detected. Anthropologic impacts, together with clonal propagation, genetic drift and geographical isolation might be the reasons which had shaped the genetic structure of this species.     

Population structure and genetic diversity of Dysosma versipellis (Berberidaceae), a rare endemic from China

Dysosma versipellis (Berberidaceae) is an endangered and endemic species in China. To provide scientific foundation for formulating conservation strategies, we sampled six extant populations of this species and assessed the levels and patterns of genetic diversity using ISSR markers (11 primers). Of 144 bands detected 57.64% were polymorphic, but on average only 20.72% were polymorphic within populations. Our results revealed a low level of intraspecific genetic diversity (at population level: H_pop = 0.082, H_B = 0.177, SI = 0.1194; at species level: H_pop = 0.207, H_B = 0.378, SI = 0.3069). A high level of genetic differentiations among populations was detected based on Nei's genetic diversity analysis (60%), AMOVA analysis (65%), and Bayesian analysis (53%). The low levels of heterozygosity and high genetic differentiation observed in D. versipellis may be the consequence of low rate of natural recruitment, clonal growth, gene drift, and habitat fragmentation. Based on this, we suggest that in situ conservation be an important and practical measure for maintaining the genetic diversity of this species. Ex situ conservation should sample from different populations across the distribution range of the species to conserve high genetic diversity.     

Genetic diversity assessment and ex situ conservation strategy of the endangered Dendrobium officinale (Orchidaceae) using new trinucleotide microsatellite markers

Dendrobium officinale (Orchidaceae) is an endangered plant species with important medicinal value. To evaluate the effectiveness of ex situ collection of D. officinale genetic diversity, we developed 15 polymorphic trinucleotide microsatellite loci of D. officinale to examine the genetic diversity and structure of three D. officinale germplasm collections comprising 120 individuals from its germplasm collection base and their respective wild populations consisting of 62 individuals from three provinces in China. The three germplasm collections showed reductions in gene diversity and average number of alleles per locus, but an increase in average number of rare alleles (frequency?≤?0.05) per locus in comparison to their wild populations. However, the differences in gene diversity between the germplasm collections and wild populations were not statistically significant. The analysis using STRUCTURE revealed evident differences in genetic composition between each germplasm collection and its wild population, probably because the D. officinale individuals with distinct genotypes in each wild population were unevenly selected for establishing its germplasm collection. For conservation management plans, we propose that D. officinale individuals with rare alleles need to be conserved with top priority, and those individuals with the most common alleles also should be concerned. The 15 new microsatellite loci may be used as a powerful tool for further evaluation and conservation of the genetic diversity of D. officinale germplasm resources.     

Genetic diversity within and among populations of the endangered species Taxus fuana (Taxaceae) from Pakistan and implications for its conservation

The West Himalayan yew, Taxus fuana Nan Li & R.R. Mill (Taxaceae), is an endangered species endemic to the Western Himalayas. An investigation of the genetic diversity of wild populations of T. fuana in Pakistan was undertaken. The genetic diversity and genetic structure was quantified using random amplified polymorphic DNA (RAPD) variation in 219 individuals of the 10 populations. Of the 32 universal primers screened 16 produced highly reproducible, clear RAPD bands. Using these primers, 193 discernible DNA fragments were generated, of which 164 (84.97%) were polymorphic. The statistical results indicated that there was a relatively low genetic diversity within populations (with percentages of polymorphic bands, PPB, ranging from 29.53 to 50.26%, with an average of 38.34% and a Nei's genetic diversity index (H_E) of 0.1165), and a high genetic differentiation among populations (G_ST = 0.5842, Φ_ST = 0.5685) within these populations. The gene flow (N_m) was low with only 0.3558.     

ISSR Analysis of the Genetic Diversity of the Endangered Species Sinopodophyllum hexandrum （Royle） Ying from Western Sichuan Province, China

Slnopodophyllum hexandrum （Royle） Ying Is an Important medicinal and endangered species. Inter-simple sequence repeats （ISSR） analysis was conducted on seven natural populations from western Slchuan Province to Investigate the genetic diversity of S. hexandrum. Leaf samples of 140 Individuals were collected. Of the 139 discernible fragments generated by 12 selected primers （among 100 primers）, 54 appeared to be polymorphlc. The percentage of polymorphlc bands （PPB） was 38.85% at the species level, and PPB within a population ranged from 7.91% to 23.74%. Low levels of genetic variation （He = 0.092, Ho = 0.142） and high levels of genetic differentiation among the populations （Gst= 62.25%） was detected on the basis of results from POPGENE and analysis of molecular variance （AMOVA）, respectively. Furthermore, the limited gene flow （Nm = 0.361） may result from biological characteristics, such as self-pollination and short distance seed dispersal. Based on the genetic and ecological Information available for S. hexandrum, we propose some appropriate strategies for the conservation of the endangered medicinal species in this region, namely rescuing and conserving the core populations for in situ conservation and sampling and preserving more populations with fewer Individuals from each population for ex situ conservation.     

Genetic diversity of the endangered species Kirengeshoma palmata (Saxifragaceae) in China

In order to investigate the levels of genetic diversity of the endangered species Kirengeshoma palmata (Saxifragaceae), four extant populations were sampled and analyzed using inter-simple sequence repeats (ISSR) markers. We expected a low genetic diversity level, but our results revealed a high level of intraspecific genetic diversity, probably resulting from this species being in a refuge during the last glaciation (at population level: P = 63.25%, A_e = 1.47, H_E = 0.26 and H_O = 0.37; at species level: P = 79.00%, A = 1.5538, H_T = 0.2586 and H_sp = 0.3104). A low level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (16.69%) and AMOVA (19.36%). Populations shared high levels of genetic identity. Insect pollination and seed dispersal by wind may have facilitated extensive gene flow and are likely responsible for this present structure of genetic variation.     

Genetic diversity and population structure of endangered endemic Paeonia jishanensis in China and conservation implications

Paeonia jishanensis, one of the most important ancestral species of cultivated tree peonies, is an endangered ornamental and medicinal plant endemic to China. A total of 236 individuals of P. jishanensis from 10 extant populations were analysed using 21 EST-SSR markers to assess their genetic diversity and population structure. Moderate genetic diversity levels (H_E = 0.340) and high genetic differentiation among populations (F_ST = 0.335) were revealed. Combining the results of STRUCTURE, PCoA and neighbour-joining analyses, the 10 populations were divided into four genetic groups that were significantly related to their geographical origins, which was further supported by hierarchical AMOVA showing the highest variation of 17.9% among groups. The Mantel test showed a significant positive correlation between genetic distance and geographic distance among populations (r = 0.873, P < 0.0001). The genetic structure of P. jishanensis may be due to limited gene flow hindered by vicariance and its breeding system: facultative vegetative reproduction. This study carries significant implications for the conservation and utilization of this endangered species.     

Genetic diversity and population structure of the medicinal orchid Gastrodia elata revealed by microsatellite analysis

The wild resources of Gastrodia elata are currently threatened with extinction due to over-harvesting because of their high medicinal value. Genetic diversity plays a key role in the survival of endangered orchid species. In this study we investigated the genetic pattern in eight microsatellite loci within eight G. elata populations from central China. Compared with the other orchids, G. elata showed a low level of genetic variation within populations (H_E = 0.356–0.622). The main factors responsible for the genetic pattern were the plant's inbreeding system due to mating within clone patches, and the genetic bottlenecks and genetic drift caused by a long-history over-collecting. The significant heterozygote deficit was detected in all the populations. The F statistics calculated by different approaches consistently revealed a clear genetic differentiation among populations, contributing about 20% of the total gene diversity. The results are discussed in relation to both in situ and ex situ conservation efforts of the species. The populations with a high level of genetic diversity or with great genetic distinction were identified, which should be a high priority for conservation managers.     

Genetic diversity of the endangered Chinese endemic herb Dayaoshania cotinifolia (Gesneriaceae) revealed by simple sequence repeat (SSR) markers

Dayaoshania cotinifolia W.T. Wang is a critically endangered perennial herb endemic to southern China. Simple sequence repeat (SSR) markers were employed to assess genetic diversity in two populations of D. cotinifolia. Eight primer pairs generated a total of 36 alleles, with a mean of 4.5 alleles per locus. The expected heterozygosity (H_e = 0.416) and observed heterozygosity (H_o = 0.508) indicated a moderate level of genetic diversity, though the genetic differentiation between the populations was low (F_st = 0.014), a result that was supported by a higher gene flow (N_m = 18.000). No severe bottleneck effect was detected in the two populations. Thus, the endangered status of this species is most likely due to anthropologic effects rather than a lack of genetic diversity. In situ conservation strategies should be promoted, and the sizes of the populations should be increased through artificial breeding.     

Genetic diversity analysis of the endangered slipper orchid Phragmipedium longifolium in Costa Rica

Phragmipedium longifolium is an endangered terrestrial orchid. In Costa Rica, these plants are found growing in small and isolated patches, some of them consisting of just four to six individuals. Information about the genetic variability within and among populations is very important for the conservation of this endangered species. A total of 160 samples were collected in six locations and analyzed with amplified fragment length polymorphism technique. The genetic diversity of P. longifolium in Costa Rica (Hw = 0.1711) is high and differentiation among sampled locations is moderate (φ_pt = 0.2013) in comparison with results of studies in some other terrestrial orchid species using the same technique. The percentage of polymorphic loci was, on average, 51.5. The analysis of molecular variance (AMOVA) indicated the main genetic variation was within sampled locations (80%), even though the variation among locations was also significant. In situ conservation is recommended because, in addition to protecting habitat and avoiding fragmentation, mycorrhizal fungi and pollinators are also protected. Close proximity between populations is required to maintain high genetic variability through a gene flow continuum. It is suggested that conservation of patches with higher genetic variability be prioritized as many are located in unprotected areas. A germplasm bank for ex situ conservation has been established in the living collection at Lankester Botanical Garden using the plants collected in this study. Finally, a search for new locations of this species is also suggested.