Genetic diversity and structure of an endangered medicinal plant species (Pilocarpus microphyllus) in eastern Amazon: implications for conservation

Few studies have evaluated the genetic status of medicinal plants exposed to commercial harvesting. Here, we examine the genetic variability of Pilocarpus microphyllus, an endemic and threatened medicinal plant species from the eastern Amazon, across its largest remaining wild population. Popularly known as jaborandi, species of Pilocarpus genus are the unique known natural source of pilocarpine, an alkaloid used to treat glaucoma and xerostomia. However, Populations of P. microphyllus has experienced a severe decline in the last decades. Using RAD sequencing, we identified a total of 5,266 neutral and independent SNPs in 277 individuals collected from the Carajás National Forest (CNF). We quantified genetic diversity and gene flow patterns and estimated the minimum number of individuals necessary to establish a germplasm bank. Our results revealed high genetic diversity and four spatially distinct clusters of P. microphyllus with substantial admixture among them. Geographic distance and temperature dissimilarity were the factors that best explained the relatedness patterns among individuals. Additionally, our findings indicate that at least 40 matrices sampled randomly from each population would be required to conserve genetic diversity in the long term. In short, P. microphyllus showed high levels of genetic diversity and an effective population size (N_E) sufficient to reduce the likelihood of extinction due to inbreeding depression. Our results indicate that diversity has been maintained despite the continuous harvesting of raw leaf material in the area over recent decades. Finally, the results provide information essential for the design of a germplasm bank to protect the endangered medicinal plant species.

     

Clonal diversity and conservation genetics of the medicinal plant Carapichea ipecacuanha (Rubiaceae)

The roots of the understorey shrub Carapichea ipecacuanha (ipecac) have medicinal properties, and the uprooting of wild plants has supplied most of the world demand for this species. Although under severe population decline, C. ipecacuanha lacks legal protection. In the wild, the aerial stems of ipecac clump together to form clusters with well-defined borders. Cluster size may range from several to hundreds of aerial stems. To investigate the extent of clonality among aerial stems in ipecac clusters, we sampled 50 wild clusters (a total of 291 aerial stems) and screened them with 89 inter-simple sequence repeat (ISSR) markers. The 291 aerial stems were grouped into 42 putative clones. The clonal groups generally consisted of aerial stems from the same cluster, and there was little or no genetic differentiation among aerial stems at the cluster level. These findings suggest that strategies designed to conserve ipecac in situ should not rely upon census data, which are based on the number of aerial stems per cluster and the number of clusters per population, because such data greatly underestimate the species effective population size and genetic diversity. Our results also indicate that this species needs protection at a federal level.     

Genetic diversity of Chinese water deer (Hydropotes inermis inermis): implications for conservation

The Chinese water deer (Hydropotes inermis inermis) is endemic to China. Historically, the species was widely distributed, but now, habitat loss and poaching have reduced its range and number drastically. In order to provide useful information for its conservation, we have investigated the genetic diversity and population structure of the Chinese water deer by analyzing the 403 bp fragment of the mitochondrial DNA (mtDNA) control region (D-loop). Eighteen different haplotypes were detected in 40 samples. Overall, Chinese water deer have a relatively high-genetic diversity compared to other rare cervid species, with a haplotype diversity of 0.923+/-0.025 and nucleotide diversity of 1.318 +/- 0.146%. No obvious phylogenetic structure among haplotypes was found for samples of different origin. An analysis of molecular variance (AMOVA) showed significant differentiation between the Zhoushan and the mainland population (F(ST)= 0.088, P < 0.001; Phi( ST ) = 0.075, P = 0.043), which suggests that exchanges of individuals between Zhoushan and the mainland should be avoided. We also recommend that a breeding center be set up for the mainland population.     

Genetic diversity of Stemona parviflora: A threatened myrmecochorous medicinal plant in China

The plant species Stemona parviflora, which is endemically distributed in Hainan and Guangdong Provinces of China, is an important constituent of Chinese traditional medicine “Baibu”. Based on AFLP analysis, at the species level, the percentage of polymorphic bands (PPB), gene diversity H, and Shannon's information index I of S. parviflora are 100%, 0.2826, and 0.4458, respectively. The genetic differentiation among populations is low (G_st = 0.0799), while the gene flow among these populations is high (N_m = 5.754). Actually, no distinguishable genetic structure is inferred according to the AFLP analysis in this study. There was no significant positive correlation between genetic distance and geographic distance either. Based on its medicinal use, seed dispersal pattern (myrmecochory), mating system, fragmentation of the natural habitat by rubber and eucalyptus planting, and the historical formation and transition of Hainan island and Guangdong provinces, we discuss the current threatened condition of the traditional medicinal plant S. parviflora. In/ex situ conservation tactics are recommended to save this important species.     

Genetic diversity and conservation and utilization of plant genetic resources

Biodiversity refers to variation within the living world, while genetic diversity represents the heritable variation within and between populations of organisms, and in the context of this paper, among plant species. This pool of genetic variation within an inter-mating population is the basis for selection as well as for plant improvement. Thus, conservation of this plant genetic diversity is essential for present and future human well-being. During recent years, there has been increasing awareness of the importance of adopting a holistic view of biodiversity, including agricultural biodiversity, conservation for sustainable utilization and development. These principles have been enshrined in the Convention on Biological Diversity and the Global Plan of Action of the Food and Agriculture Organization of the United Nations. The emphasis is now to understand the distribution and extent of genetic diversity available to humans in plant species, so that the genetic diversity can be safely conserved and efficiently used. It is generally recognized that plant genetic diversity changes in time and space. The extent and distribution of genetic diversity in a plant species depends on its evolution and breeding system, ecological and geographical factors, past bottlenecks, and often by many human factors. Much of the large amount of diversity of a species may be found within individual populations, or partitioned among a number of different populations.A better understanding of genetic diversity and its distribution is essential for its conservation and use. It will help us in determining what to conserve as well as where to conserve, and will improve our understanding of the taxonomy and origin and evolution of plant species of interest. Knowledge of both these topics is essential for collecting and use of any plant species and its wild relatives. In order to mange conserved germplasm better, there is also a need to understand the genetic diversity that is present in collections. This will help us to rationalize collections and develop and adopt better protocols for regeneration of germplasm seed. Through improved characterization and development of core collections based on genetic diversity information, it will be possible to exploit the available resources in more valuable ways.     

青藏高原药用植物分布格局及保护优先区

青藏高原拥有丰富的药用植物资源, 但目前人们缺乏对其多样性分布格局的清晰认识以及人类活动对药用植物资源影响的评估。本研究收集整理了青藏高原地区254种药用植物分布及人类活动的数据, 分析了当前青藏高原地区药用植物分布格局及其面临的威胁, 并划定出当前需要保护的优先区域。研究结果显示, 青藏高原药用植物的多样性和特有性主要集中在东部和东南部地区, 且较强的人类活动影响力与较高的药用植物多样性和特有性重叠, 这表明人类活动对药用植物多样性和特有性有着显著的影响。进一步结合生物和人类活动两方面因素的算法, 划定了保护优先区, 包括云南西北部、四川西南部和西部、青海东部及西藏中部, 这表明青藏高原现有的保护区存在大量保护空缺。基于此, 本研究提出了包括完善法律法规及政策监管, 增设保护区, 补充和完善保护植物名录, 加强科普宣传, 加强药用植物种质资源的收集、保存与开发的保护青藏高原药用植物资源的建议, 为今后保护政策的制定和保护区划定提供依据, 也借此宣传青藏高原生态保护问题, 唤起民众的保护关注。     

Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae)

Magnolia officinalis subsp. biloba, a traditional Chinese medicinal plant, experienced severe declines in the number of populations and the number of individuals in the late 20th century due to the widespread harvest of the subspecies. A large-scale cultivation program was initiated and cultivated populations rapidly recovered the loss in individual plant numbers, but wild populations remained small as a consequence of cutting. In this study, the levels of genetic variation and genetic structure of seven wild populations and five domestic populations of M. officinalis subsp. biloba were estimated employing an AFLP methodology. The plant exhibited a relatively high level of intra-population genetic diversity (h = 0.208 and H _j = 0.268). The cultivated populations maintained approximately 95% of the variation exhibited in wild populations, indicating a slight genetic bottleneck in the cultivated populations. The analysis of genetic differentiation revealed that most of the AFLP diversity resided within populations both for the wild group (78.22%) and the cultivated group (85.92%). Genetic differentiation among populations in the wild group was significant (F _ST = 0.1092, P < 0.005), suggesting wild population level genetic structure. Principal coordinates analysis (PCO) did not discern among wild and cultivated populations, indicating that alleles from the wild population were maintained in the cultivated gene pool. Results from the present study provide important baseline data for effectively conserving the genetic resources of this medicinal subspecies.     

Genetic variation among wild and cultivated populations of the Chinese medicinal plant Coptis chinensis (Ranunculaceae)

To examine if the cultivation process has reduced the genetic variation of modern cultivars of the traditional Chinese medicinal plant, Coptis chinensis, the levels and distribution of genetic variation was investigated using ISSR markers. A total of 214 C. chinensis individuals from seven wild and three cultivated populations were included in the study. Seven ISSR primers were used and a total of 91 DNA fragments were scored. The levels of genetic diversity in cultivated populations were similar as those in wild populations (mean PPL = 65.2% versus PPL = 52.4%, mean H = 0.159 versus H = 0.153 and mean I = 0.255 versus I = 0.237), suggesting that cultivation did not seriously influence genetic variation of present-day cultivated populations. Neighbour-joining cluster analysis showed that wild populations and cultivated populations were not separated into two groups. The coefficient of genetic differentiation between a cultivar and its wild progenitor was 0.066 (G(st)), which was in good accordance with the result by amova analysis (10.9% of total genetic variation resided on the two groups), indicating that cultivated populations were not genetically differentiated from wild progenitors. For the seven wild populations, a significant genetic differentiation among populations was found using amova analysis (45.9% of total genetic variation resided among populations). A number of causes, including genetic drift and inbreeding in the small and isolated wild populations, the relative limited gene flow between wild populations (N(m) = 0.590), and high gene flow between cultivars and their wild progenitors (N(m) = 7.116), might have led to the observed genetic profiles of C. chinensis.     

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.     

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.     

桑黄真菌分子鉴定及遗传多样性分析

药用真菌桑黄具有明显的抗肿瘤、抗氧化、增强免疫等药理活性,但研究者对其基源还没有达成共识,多种Phellinus属真菌被当作桑黄入药使用。采用rDNA ITS序列分析技术,对桑黄真菌进行分子鉴定及遗传多样性分析。通过rDNA ITS序列分析,成功鉴定出一份混淆样品(Phellinus spp-04),并将中国主要使用的桑黄真菌明确鉴定为P.baumii和P.linteus两种,未检测到P.igniarius的使用。依据rDNA ITS序列计算遗传距离并构建系统发育树,结果表明3种主要桑黄真菌存在明显的遗传分化,在系统发育树中明确聚为3个独立菌种类群。在3种桑黄真菌rDNA ITS序列中,存在颠换、转换及插入/缺失3种类型的变异位点,分别在P.linteus、P.baumii和P.igniarius中鉴定出9、9、8种rDNA ITS单倍型序列,不同单倍型菌种间遗传分歧度变化表现出明显的物种差异性。     

Genetic diversity of Lavandula multifida L. (Lamiaceae) in Tunisia: implication for conservation

The genetic variation within and among eight Tunisian natural populations of Lavandula multifida L., from different bioclimatic zones was assessed using random amplified polymorphic DNA (RAPDs). Of a total of 97 generated bands from seven selected primers, 84 bands were polymorphic. The genetic diversity within a population was high and varied according to the populations (0.308 < H’ < 0.459) without relationships to altitudes or pluviothermic indices of sites. The genetic differentiation among populations was high (G_ST = 0.395 and Φ_ST = 0.318). All population pairs were significantly differentiated. Among populations, within ecological groups genetic structure was high (0.219); whilst among them it was low (Φ_CT = 0.049; P < 0.05). The correlation between Φ_ST and geographic distance matrices among pairs of populations was not significant, suggesting that genetic connectivity between populations has a stochastic component at all spatial scales. The neighbour‐joining cluster analysis showed that individuals from each population clustered together. UPGMA cluster analysis showed that population groupings are not strictly in accordance with bioclimates or geographic location. The genetic differentiation in L. multifida could have occurred at local scales because of genetic drift. Efforts should be made to protect all populations. The maintenance of substantial population size should be initiated via fencing and controlling collection to restore the regeneration of populations.     

Genetic diversity and differentiation in the critically endangered orchid (Amitostigma hemipilioides): implications for conservation

Amitostigma hemipilioides is an endangered terrestrial orchid endemic to China. In the Southwestern China, the species is found restricted growing in the karst limestone or rock in the edge of farmland, moist hillsides or river. In the present study, the genetic diversity and differentiation was estimated within and among habitats, populations and groups of this species by ISSR markers. Using 13 polymorphic primers, an intermediate level of genetic diversity was found at the species level and population level with the percentage of polymorphic bands (P) of 64.7 and 50.9 %, Shannon index of diversity (I) of 0.3873 and 0.2949, respectively. The analysis of molecular variance (AMOVA) showed that the high level of population differentiation was presented with 45.63 % relative to the total genetic variation residing among eight populations. It was noteworthy that as much as 69.17 % of the total diversity was most likely attributed to the difference among the populations in fragmentation habitat conditions, while 16.32 % of the total diversity could be attributed to the difference among the populations in stable and favorable habitat conditions. The in situ conservation is a top strategy, thus the mycorrhizal fungi and pollinators are protected by protecting habitat and avoiding fragmentation which is helpful to the cycle of this endangered orchid species and recovery of its wild populations. In addition, to maintaining the germplasm bank of this species, the ex situ conservation by habitat simulation and reintroduction has been considered in the living collection at Kaili University ecological garden and Yuntai Mountain scenic spot using the plants collected in this study.     

Evaluation of traditional medicinal plant,Cissus setosa Roxb. (Vitaceae) for antiulcer property

Cissus setosa is an indigenous medicinal herb commonly used for the treatment of gastro ulcers. In the current investigation the aerial methanolic extract of C. setosa was investigated for their antiulcer activity using pylorus ligation and ethanol in experimental rats. The extract was administered at the doses of 200 and 400 mg/kg b.w. orally for 3 days. However, higher dose of the extract subsequently reduced gastric ulcer induced aberrations by pylorus ligation (70.05%) and ethanol (78.16%) as judged by their altered biochemical parameters such as free acidity, total acidity, total carbohydrate, total protein and pepsin activity. Furthermore, macroscopic examination of rat’s stomach also showed that the pretreatment with methanolic extract notably lowered the pylorus ligation and ethanol induced ulcers. As perceived in the present study, evidently, our findings basically supports the potency of the methanol extracts of C. setosa to treat gastrointestinal related disorders, thus lends pharmacological credence to the suggested folklore use.     

Genetic diversity of captive binturongs (Arctictis binturong, Viverridae, Carnivora): implications for conservation

The binturong Arctictis binturong is a threatened carnivore (Mammalia) that ranges throughout the forests of South-east Asia. This study evaluates the genetic diversity of captive binturong populations in European zoos and attempts to assess their geographic origin. We sequenced the hypervariable region 1 of the mitochondrial control region of 56 binturongs, among which 20 had a known geographic origin. We showed that at least two distinct geographic clades exist and were able to assess the geographic clade to which captive individuals belong. Moreover, a low genetic diversity was observed among the captive population of European zoos. Although our results are preliminary, zoo managers should consider the evolutionary significant units identified by this study, and which correspond to recognized sub-species, when planning binturong reproduction programs.     

Genetic diversity, phylogeny and conservation of the Javan rhinoceros (Rhinoceros sondaicus)

With a total population of less than 60 individuals limited to two locations, the Javan rhinoceros is perhaps the most endangered large mammal on earth. Although species specific information is crucial to its conservation, its precarious status, habitat inaccessibility, and behavioral adaptations pose major obstacles to its study. Here we report on the first genetic analysis of the two extant populations, in Ujung Kulon, Indonesia, and Cat Tien, Vietnam, and discuss their conservation. As its critically endangered status precluded invasive sampling, we extracted DNA from dung, amplifying and sequencing segments of the mtDNA 12S rRNA gene and the non-coding D-loop. Divergence between Javan rhinos from Ujung Kulon and Cat Tien was similar to that between recognized subspecies of African rhinos, and exceeded that between Sumatran rhinos. The Ujung Kulon and Cat Tien populations represent separate Evolutionary Significant Units, advocating independent management. However, given the precariousness of the Cat Tien population, demographic considerations may override genetic issues in the short term. Genetic diversity of Javan rhinos was low and population expansion in the immediate future will be critical for its survival.