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 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 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 and population structure of the Asian green mussel Perna viridis in South China Sea based on microsatellite markers

The green mussel, Perna viridis is ecologically and economically important in the coastal region of the South China Sea. Determining its population genetic structure at this fine geographic scale will help sustainable management of natural stocks. In this study, we examined the genetic diversity and population genetic structure of P. viridis from four locations in the South China Sea (n = 45–48) using nine microsatellite loci. The results showed moderate levels of genetic diversity in all four samples (mean A = 13.222–14.000, mean A_e = 7.092–7.571, mean A_r = 12.894–13.746, mean H_o = 0.596–0.656, mean H_e = 0.690–0.733) and a large effective population size estimate for the pooled sample (total N_e estimates = infinity, 95% CI = 1869.0-infinity). We did not detect any sign of recent bottleneck events in P. viridis populations in the South China Sea. The conventional and a model-based analysis reveal low, non-significant genetic divergence among the four samples (F_ST = − 0.001–0.005, P > 0.05/6). The results obtained from this study can provide valuable genetic information for the conservation and fishery management of P. viridis by retaining the high N_e estimates.     

High genetic diversity in Taihangia rupestris Yu et Li,a rare cliff herb endemic to China,based on inter-simple sequence repeat markers

Taihangia rupestris Yu et Li is an endangered perennial herb endemic to China. Besides being at an important systematic position in the tribe Dryadeae, it also is a valuable wild flower resource. Inter-simple sequence repeat (ISSR) markers were employed to estimate the genetic diversity and genetic differentiation of ten populations. Relatively high genetic diversity was detected at species levels (PPB = 80.43; h = 0.2479; I = 0.3785). Most of the variation (73.51%) occurred within populations, and 26.49% was found among populations; Nei’s differentiation coefficient (Gst = 0.2435) supported this result. The Mantel test revealed a significantly positive correlation between geographical distance and genetic distance (r = 0.611, P < 0.001). The levels and patterns of genetic diversity of T. rupestris were assumed to result from its ancient origin and mixed reproductive modes. Based on these results, both ex situ and in situ conservation are 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.     

Analysis of genetic diversity in Aconitum kongboense L. revealed by AFLP markers

The systematic evaluation of the molecular diversity encompassed in Aconitum kongboense L. inbreds or parental lines offers an efficient means of exploiting the heterosis in A. kongboense as well as for management of biodiversity. An excellent and novel DNA-based molecular, amplified fragment length polymorphisms (AFLPs) markers, was firstly used to analysis the genetic diversity in A. kongboense genotypes. Out of 256 primers screened, a total of ten combinations successfully produced scorable, clear, reproducible and relatively high polymorphism bands, 64.12% of which were polymorphic. The values of number of polymorphic loci (NPL), percentage of polymorphic loci (PPB), observed number of alleles (Na) and effective number of alleles (Ne) were highest in population 3 (NPL = 77, PPB = 68.75%, Na = 1.688 ± 0.466, Ne = 1.412 ± 0.397) and lowest in population 2 (NPL = 57, PPB = 50.89%, Na = 1.509 ± 0.502, Ne = 1.273 ± 0.340). In addition, Jaccard's similarity coefficients among different populations varied from 0.45 to 1.00 with an average of 0.55. The data collected will contribute to identification, rational exploitation and conservation of germplasms of A. kongboense, and potentially useful to aid its breeding.     

Genetic diversity and population structure in Vallisneria spinulosa (Hydrocharitaceae)

Vallisneria spinulosa is a dominant submerged macrophyte in lakes of the middle–lower reaches of the Yangtze River. Allozyme variation, clonal diversity and population genetic structure were investigated for a total of 396 individuals sampled from 10 extant populations. V. spinulosa maintained high levels of genetic variation both at the species (P = 46.2, A = 1.69, H_e = 0.23) and at the population level (P = 46.2, A = 1.58, H_e = 0.21). Although aquatic macrophytes commonly exhibit low genetic variation within populations, the obligately outcrossing mating system of V. spinulosa and pervasive gene flow likely account for the high levels of diversity maintained within populations. All V. spinulosa populations contained high clonal diversity with a mean proportion of distinguishable genotypes of 0.57 and a mean Simpson's diversity index of 0.95, indicating that populations were founded sexually or that successful seedling recruitment occurred after initial colonization. Partitioning of genetic diversity revealed a surprisingly low population differentiation (G_ST = 0.06) as compared to other hydrophilous angiosperms. No evidence of isolation-by-distance was found (r = 0.056, P = 0.312), suggesting that gene flow was not restricted geographically. The UPGMA cluster analysis revealed that several widely separated populations grouped together, suggesting long-distance gene flow among populations. The high vagility of V. spinulosa and extensive hydrologic connectivity among populations have facilitated long-distance gene flow and resulted in the pattern of population genetic structure in V. spinulosa.     

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 variation and effective population size in isolated populations of coastal cutthroat trout

Following glacial recession in southeast Alaska, waterfalls created by isostatic rebound have isolated numerous replicate populations of coastal cutthroat trout (Oncorhynchus clarkii clarkii) in short coastal streams. These replicate isolated populations offer an unusual opportunity to examine factors associated with the maintenance of genetic diversity. We used eight microsatellites to examine genetic variation within and differentiation among 12 population pairs sampled from above and below these natural migration barriers. Geological evidence indicated that the above-barrier populations have been isolated for 8,000–12,500 years. Genetic differentiation among below-barrier populations (F _ST = 0.10, 95% C.I. 0.08–0.12) was similar to a previous study of more southern populations of this species. Above-barrier populations were highly differentiated from adjacent below-barrier populations (mean pairwise F _ST = 0.28; SD 0.18) and multiple lines of evidence were consistent with asymmetric downstream gene flow that varied among streams. Each above-barrier population had reduced within-population genetic variation when compared to the adjacent below-barrier population. Within-population genetic diversity was significantly correlated with the amount of available habitat in above-barrier sites. Increased genetic differentiation of above-barrier populations with lower genetic diversity suggests that genetic drift has been the primary cause of genetic divergence. Long-term estimates of N _e based on loss of heterozygosity over the time since isolation were large (3,170; range 1,077–7,606) and established an upper limit for N _e if drift were the only evolutionary process responsible for loss of genetic diversity. However, it is likely that a combination of mutation, selection, and gene flow have also contributed to the genetic diversity of above-barrier populations. Contemporary above-barrier N _e estimates were much smaller than long-term N _e estimates, not correlated with within-population genetic diversity, and not consistent with the amount of genetic variation retained, given the approximate 10,000-year period of isolation. The populations isolated by waterfalls in this study that occur in larger stream networks have retained substantial genetic variation, which suggests that the amount of habitat in headwater streams is an important consideration for maintaining the evolutionary potential of isolated populations.     

The return of the frogs: The importance of habitat refugia in maintaining diversity during a disease outbreak

Recent decades have seen the emergence and spread of numerous infectious diseases, often with severe negative consequences for wildlife populations. Nevertheless, many populations survive the initial outbreaks, and even undergo recoveries. Unfortunately, the long‐term effects of these outbreaks on host population genetics are poorly understood; to increase this understanding, we examined the population genetics of two species of rainforest frogs (Litoria nannotis and Litoria serrata) that have largely recovered from a chytridiomycosis outbreak at two national parks in the Wet Tropics of northern Australia. At the wetter, northern park there was little evidence of decreased genetic diversity in either species, and all of the sampled sites had high minor allele frequencies (mean MAF = 0.230–0.235), high heterozygosity (0.318–0.325), and few monomorphic markers (1.4%–4.0%); however, some recovered L. nannotis populations had low N_e values (59.3–683.8) compared to populations that did not decline during the outbreak (1,537.4–1,756.5). At the drier, southern park, both species exhibited lower diversity (mean MAF = 0.084–0.180; heterozygosity = 0.126–0.257; monomorphic markers = 3.7%–43.5%; N_e = 18.4–676.1). The diversity patterns in this park matched habitat patterns, with both species having higher diversity levels and fewer closely related individuals at sites with higher quality habitat. These patterns were more pronounced for L. nannotis, which has lower dispersal rates than L. serrata. These results suggest that refugia with high quality habitat are important for retaining genetic diversity during disease outbreaks, and that gene flow following disease outbreaks is important for re‐establishing diversity in populations where it was reduced.     

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 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.     

Identification,development, and application of 12 polymorphic EST-SSR markers for an endemic Chinese walnut (Juglans cathayensis L.) using next-generation sequencing technology

The Chinese walnut (Juglans cathayensis L.), valued for both its nut and wood, is an ecologically important tree species endemic temperate southern China. Investigation of the genetic diversity of Chinese walnut has been limited to natural population genetics and genetic germplasm resources. Here, we describe the development of 12 polymorphic microsatellite markers using next-generation sequencing to screen 96 Chinese walnut individuals collected from 11 natural populations. The number of alleles per locus ranged from 5 to 12. The observed heterozygosity (0.288–0.748) overlapped well with the expected heterozygosity (0.337–0.751). This species has high genetic diversity and gene flow among different populations (F_ST = 0.075, Nm = 3.088). These markers will be useful for future studies on population genetic structure, evolutionary ecology, and genetic breeding of this walnut tree or other Juglans species.     

Influence of translocation strategy and mating system on the genetic structure of a newly established population of island ptarmigan

Island populations and populations established by reintroductions are prone to extinction, in part because they are vulnerable to deterministic and stochastic phenomena associated with geographic isolation and small population size. As population size declines, reduced genetic diversity can result in decreased fitness and reduced adaptive potential, which may hinder short- or long-term population viability. We used 32 microsatellite markers to investigate the conservation genetics of a newly established population of Evermann’s Rock Ptarmigan (Lagopus muta evermanni) at Agattu Island, in the western Aleutian Archipelago, Alaska. We found low genetic diversity (observed heterozygosity = 0.41, allelic richness = 2.2) and a small effective population size (N _e  = 28.6), but a relatively large N _e /N ratio = 0.55, which was attributed to multiple paternity in 80% of the broods and low reproductive skew among males (λ = 0.29). Moreover, successful breeding pairs were less related to each other than random male–female pairs. For conservation efforts based on reintroductions, a mating system with high rates of multiple paternity may facilitate retention of genetic diversity, thereby reducing the potential for inbreeding in small or isolated populations. Our results underscore the importance of quantifying genetic diversity and understanding the breeding behavior of translocated populations.     

Genetic differentiation in endangered Gynostemma pentaphyllum (Thunb.) Makino based on ISSR polymorphism and its implications for conservation

Studies were performed to investigate the genetic variation of 14 natural populations of Gynostemma pentaphyllum (Thunb.) Makino, an outcrossing clonal plant species in China, using inter-simple sequence repeat (ISSR) markers. Fourteen selected primers were used to amplify DNA samples from 140 individuals, and totally 194 loci were detected. The percentage of polymorphic bands (PPBs) showed that the genetic diversity was pretty high at the species level (PPB = 96.39%) but quite low at the population level (PPB = 1.03–25.26%). Shannon's information index (I) and Nei's gene diversity (h) displayed a similar trend to PPB. According to the hierarchical analysis of molecular variance (AMOVA) and Nei's analysis of gene diversity, the percentages of genetic variation among populations were 88.66 and 88.94%, respectively, indicating a high level of inter-population genetic differentiation. The low levels of genetic diversity within populations and high genetic differentiation among populations were assumed to result from the limited gene flow, the clonal nature and genetic drift. Based on the genetic data, effective conservation strategies were proposed for conserving this traditional Chinese medicinal herb. Concerning the management of G. pentaphyllum, we suggested that in situ conservation be an important and practical measure for maintaining the genetic diversity and that a possibly maximum number of populations be conserved. Populations EMS and HLT, in which particularly low levels of genetic variation were characterized, should be given the priority for ex situ conservation.     

Genetic diversity of Shiraia bambusicola from East China assessed using ISSR markers

To understand the genetic differentiation of the medicinal fungus Shiraia bambusicola, the genetic diversity of 107 individuals from eight populations collected from Jiangsu, Anhui and Zhejiang provinces were studied using inter-simple sequence repeat (ISSR) analysis. The results revealed that the 11 employed primers produced a total of 241 ISSR loci, of which 240 loci (PPB = 99.6%) were polymorphic. Both Nei's gene diversity indexes and Shannon's gene diversity indexes showed that the genetic differentiation of S. bambusicola primarily occurred within the populations. AMOVA revealed that the variation among populations was 40.0%, and the variation within populations was 60.0%. The results of an unweighted pair group method arithmetic average (UPGMA) analysis and a principal coordinate analysis (PCA) revealed that the populations with minimal geographic separations frequently exhibited regional characteristics. These findings revealed that the relationship between sibship and geographical distribution was intensive.     

A molecular approach to understand the riddle of the invasive success of the tarantula,Brachypelma vagans,on Cozumel Island,Mexico

Invasive populations typically demonstrate genetic isolation which results in a loss of genetic diversity and a reduction in invasion success. This study focused on the genetic population of a successful invasive species of tarantula. Individuals were sampled in two mainland localities of the Yucatan Peninsula (Zoh-Laguna and Raudales), in addition to two island localities (El Cedral and Rancho Guadalupe on Cozumel Island). All populations present high genetic diversity (mean: H_e = 0.23, P = 99%), with significant differences between the Raudales and Rancho Guadalupe localities. The AMOVA analysis revealed a significant population structure (14.5% variation among populations), consistent with the gene differentiation coefficient (G_ST = 0.21), and spatial analysis of population structure. Our results suggested that the original introduced population did not suffer a loss of genetic diversity during establishment on the island, possibly a result of different biological conditions. Population structure analysis leads us to suggest that one island population is similar to the original genetic profile, whereas the genotypic profile of the other island population reflects recent introductions from the mainland. We identified a potential risk of extinction for one local mainland population, suggesting that this species may be a successful invader in a new environment but endangered in some parts of its natural area.     

The value of small habitat islands for the conservation of genetic variability in a steppe grass species

The habitat loss and fragmentation due to agricultural land-conversion affected the steppe throughout its range. In Ukraine, 95% of steppe was destroyed in the last two centuries. Remaining populations are confined to few refuges, like nature reserves, loess ravines, and kurgans (small burial mounds), the latter being often subject to destruction by archeological excavations.Stipa capillata L. is a typical grass species of Eurasian steppes and extrazonal dry grasslands, that was previously used as a model species in studies on steppe ecology. The aim of our research was to assess genetic diversity of S. capillata populations within different types of steppe refuges (loess ravines, biosphere reserve, kurgan) and to evaluate the value of the latter group for the preservation of genetic diversity in the study species.We assessed genetic diversity of 266 individuals from 15 populations (nine from kurgans, three from loess ravines and three from Askania-Nova Biosphere Reserve) with eight Universal Rice Primers (URPs).Studied populations showed high intra-population variability (I: 0.262–0.419, PPB: 52.08–82.64%). Populations from kurgans showed higher genetic differentiation (Φ_ST = 0.247) than those from loess ravines (Φ_ST = 0.120) and the biosphere reserve (Φ_ST = 0.142). Although the diversity metrics were to a small extent lower for populations from kurgans than from larger refugia we conclude that all studied populations of the species still preserve high genetic variability and are valuable for protection. To what extent this pattern holds true under continuous fragmentation in the future must be carefully monitored.     

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.