Capturing,protecting and restoring plant diversity in the UK: RBG Kew and the Millennium Seed Bank

Ex situ seed banking is a practical and cost-effective means of preserving wild plant diversity and a crucial complement to the in situ conservation and restoration of species and habitats. As pressures on the natural environment have grown, so has the call for seed banks to provide scientifically-robust,practical solutions to seed-related problems in nature conservation, from single-species recovery and reintroduction to the restoration of complex, dynamic communities at the largest scales. In this paper, we discuss how the Royal Botanic Gardens, Kew and its Millennium Seed Bank have responded to this call in the United Kingdom. We demonstrate that banked seed collections can provide a range of otherwiseunavailable, high quality, known-origin, genetically-diverse biological materials. The data, expertise and specialist facilities that accompany these collections are also valuable, helping overcome constraints to the collection, production and effective use of native seed. Challenges remain-to ensure ex situ collections protect the species and genetic diversity that will enable plants to adapt to a changing environment, and to find new ways for seed banks to mobilise their resources at a landscape scale.     

Conservation genetics of an ex situ population of <Emphasis Type="Italic">Primula reinii</Emphasis> var. <Emphasis Type="Italic">rhodotricha</Emphasis>, an endangered primrose endemic to Japan on a limestone mountain

Primula reinii var. rhodotricha is a perennial herb endemic to the limestone slope of Mt. Buko, located approximately 50 km northwest of central Tokyo, Japan. In recent years, its natural population size has decreased markedly due to limestone mining, and this species has been assigned to the ‘Critically Endangered (CR)’ category on the latest Japanese Red List. Although a remnant population of this species has been protected by a mining company outside their historical distribution range on Mt. Buko, the ex situ conservation of this endangered plant has been difficult because of insufficient low seed production. The genetic status of ex situ P. reinii var. rhodotricha and related species were investigated to develop an effective conservation plan for this species. Microsatellite analysis indicated that the ex situ population harbors lower genetic diversity than sister taxa, providing molecular evidence for the recent critical status designation of the ex situ population, whereas the presence of rare alleles may imply further potential for seed reproduction by outcrossing. Therefore, an appropriate propagation strategy that considers genetic diversity is needed for restoration and recovery of this critically endangered ex situ primrose population.     

Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP

Dendrobium officinale is a critically endangered perennial herb endemic to China. Determining the levels of genetic diversity and patterns of population genetic structure of this species would assist in its conservation and management. Data of 12 populations were used to assess its genetic diversity and population structure, employing the method of amplified fragment length polymorphism (AFLP). A high level of genetic diversity was detected (H (E) = 0.269) with POPGENE. As revealed by AMOVA analysis, there was moderate variation between pairs of populations with Phi(ST) values ranging from 0.047 to 0.578 and on average 26.97% of the genetic variation occurred among populations. Three main clusters were shown in UPGMA dendrogram using TFPGA, which is consistent with the result of principal coordinate ananlysis (PCO) using NTSYS. Keeping a stable environment is critical for the in situ conservation and management of this rare and endangered plant, and for ex situ conservation it is important to design an integrated germplasm bank.     

On the horns of a dilemma: molecular approaches refine ex situ conservation in crisis

Seven years into this new millennium, species and habitat loss continue at an accelerated rate. While there have been individual examples of conservation success, the trend towards catastrophic loss of biological diversity persists. If we are to be successful in saving even a handful of critically endangered species, it is clear that they will need to be intensively managed using a variety of in situ and ex situ approaches. The highest profile ex situ conservation strategy is captive breeding. Although its relative role in an overall conservation management plan varies, captive breeding may present the only viable option for propagating the future of a species once rendered extinct in the wild. The study of Iyengar et al. in this issue of Molecular Ecology on one such species, the scimitar-horned oryx (Oryx dammah), represents an important contribution to ex situ conservation, demonstrating how critical insights into demographic history and population genetic structure obtained using molecular approaches may significantly contribute to captive breeding and reintroduction strategies.     

Strategies for establishing and using genome resource banks to protect genetic diversity in conservation breeding programs

Genome resource banks (GRBs) have the potential to preserve the genetic diversity of a species over time, yet they are rarely utilized as effective components of conservation breeding programs. Advances have been made in reproductive biology, collection and storage techniques, and use of stored gametes for achieving successful reproduction, but there are few guidelines for integrating GRBs into established breeding programs. Here we present basic guidelines, focusing on strategies for the collection, maintenance, and use of semen GRBs for protecting genetic diversity. These guidelines should be applied in the context of the specific purposes and roles of a breeding program's GRB, which will differ among species depending on vulnerability to loss and the status of rescue and conservation efforts. We recommend establishing up to three types of collections: (1) a National Reserve to preserve a species' genetic diversity, to be used only as a last resort; (2) a Savings Account to be used periodically to invigorate a genetically depauperate population; and (3) a Checking Account to be used as a regular part of the breeding program. We present methods for identifying donors to maximize genetic diversity in a GRB, as well as strategies for maintaining and optimally using GRBs.     

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.     

Conservation genetics of the endangered endemic Hawaiian genus Brighamia (Campanulaceae)

The endemic Hawaiian genus Brighamia (Campanulaceae) comprises two federally endangered, morphologically similar species, B. insignis from Kaua`i and Ni`ihau and B. rockii from Moloka`i. To assist the design of conservation management programs for these taxa, isozyme analyses were performed to assess the levels of genetic diversity at the population and species levels, including comparisons within and among seven natural populations and one ex situ collection each of B. insignis and B. rockii. Our sampling (N = 80) represents ~41% of all known individuals in the wild. Isozyme analyses revealed levels of genetic variation comparable to those reported for other Hawaiian flowering plant taxa but low levels of genetic variation at the population and species levels when compared to flowering plants in general. Ex situ individuals (N = 61) were genetically representative of natural populations and hence may appropriately serve as stock for population augmentations. The two morphologically similar Brighamia species were highly distinct genetically. The combination of morphological and ecological similarity with allozymic dissimilarity observed in Brighamia is unique among the Hawaiian taxa studied to date.     

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.     

注意植物迁地保护中的遗传风险

稀有濒危植物迁地保护的长期目标之一就是要保护物种的遗传多样性和进化潜力。介绍了稀有濒危植物在植物园迁地保护过程中存在的一系列遗传风险。由于引种或取样的不足,容易导致被保存的物种缺乏足够的遗传代表性;盲目的引种、不合理的定植以及材料的来源不清则会导致稀有濒危植物的遗传混杂、近交衰退或杂交衰退;人为选择和生长环境的改变也容易造成濒危物种对迁地保护的遗传适应。在实际的迁地保护工作中,这些遗传风险往往被忽视。植物迁地保护中遗传风险将严重影响稀有濒危物种的回归和恢复。植物园应当重视濒危植物的遗传管理,以降低或避免迁地保护中的遗传风险。Abstract: Conserving genetic diversity of rare and endangered species and their evolutionary potential is one of the long-term goals of ex-situ conservation. Some potential genetic risks in ex-situ conservation in botanical gardens are presented. The preserved species may lack genetic representativity because of poor sampling. Inappropriate plantations, inadequate records and unclear kinships jeopardize endangered species to genetic confusion, inbreeding depression or outbreeding depression. Artificial selection and habitat conversion also potentially result endangered plants in adapting to ex-situ conservation, which had been usually overlooked. All the genetic risks can decrease the success of reintroduction and recovery. Therefore, appropriate genetic management should be carried out in botanical gardens to decrease or avoid genetic risks in ex-situ conservation.     

Looking back to go forward: genetics informs future management of captive and reintroduced populations of the black-footed rock-wallaby <Emphasis Type="Italic">Petrogale lateralis</Emphasis>

Active management is essential to the survival of many threatened species globally. Captive breeding programmes can play an important role in facilitating the supplementation, translocation and reintroduction of wild populations. However, understanding the genetic dynamics within and among wild and captive populations is crucial to the planning and implementation of ex situ management, as adaptive potential is, in part, driven by genetic diversity. Here, we use 14 microsatellite loci and mitochondrial Control Region sequence to examine the population genetics of both wild populations and captive colonies of the endangered warru (the MacDonnell Ranges race of the black-footed rock-wallaby Petrogale lateralis) in central Australia, to understand how historical evolutionary processes have shaped current diversity and ensure effective ex situ management. Whilst microsatellite data reveal significant contemporary differentiation amongst remnant warru populations, evidence of contemporary dispersal and relatively weak isolation by distance, as well as a lack of phylogeographic structure suggests historical connectivity. Genetic diversity within current captive populations is lower than in the wild source populations. Based on our genetic data and ecological observations, we predict outbreeding depression is unlikely and hence make the recommendation that captive populations be managed as one genetic group. This will increase genetic diversity within the captive population and as a result increase the adaptive potential of reintroduced populations. We also identify a new site in the Musgrave Ranges which contains unique alleles but also connectivity with a population 6 km away. This novel genetic diversity could be used as a future source for supplementation.     

Developing biological resource banks as a supporting tool for wildlife reproduction and conservation The Iberian lynx bank as a model for other endangered species

This work presents a Biological Resource Bank generated as a complementary supporting tool for the reproduction and the in situ and ex situ conservation of the Iberian lynx. In its design we prioritized the preservation of a maximum of the current genetic and biological diversity of the population, and the harmless collection of the samples. To provide future reproductive opportunities through any possible technique, we processed and cryopreserved germinal cells and tissues from dead animals, 7 males and 6 females, as well as somatic cells and tissues from 69 different individuals. This somatic cell reserve reflects a very important fraction of the population biodiversity which, furthermore, will allow the development of a wide variety of studies that can be easily extrapolated to the majority of the population. We have developed a new non-destructive method to isolate cells with stem-cell-like properties. If considered convenient in the future, and after proper research, such cells could permit therapeutic applications and perhaps be a good source to be used in somatic cell nuclear transfer. Samples of whole blood and its derivatives, hairs, urine and feces from many different individuals were also preserved. Proper storage of such samples is required to allow epidemiological studies to be performed for the testing of different etiological hypotheses or, in general, to develop any bio-sanitary study to improve conservation strategies within the natural habitat. This work describes the main aspects involved in the practical implementation of the Iberian lynx Biological Resource Bank, as a model that could be useful for the development of similar banks for other endangered species.     

Conservation genetic assessment of four plant species in a small replica of a steppe ecosystem &gt;30 years after establishment

To counter species loss living ex situ collections in botanic gardens became important elements of robust conservation programs. Several limitations, problems, and risks associated with living ex situ collections have been reported such as appropriate cultivation management to maintain genetic diversity and stochastic effects in small isolated populations in artificial habitats. However, not all small and isolated populations exhibit these predicted genetic changes. In a multi-species in situ/ex situ comparison of sand dune steppe- and grassland vegetation >30 years after the ex situ population establishment, we compared four different species’ population genetic diversities (Alyssum montanum ssp. gmelinii, Gypsophila fastigiata, Helianthemum nummularium ssp. obscurum, Onosma arenaria) by means of ISSR. We observed different species-specific genetic responses to quite similar abiotic selective forces concerning different neutral genetic diversities of wild versus botanic garden populations. The genetic divergence was kept relatively low in two of the four investigated species between the model steppe plant community within the botanic garden where human interference was kept at a minimum and the wild population. However, the moderate genetic divergence of the two other species kept under the same conditions highlights the importance of species-specific intrinsic responses and stochastic effects to ecosystem changes and provides data on population genetic dynamics in small and isolated populations. This contributes to further improve recommendations on how to best conserve endangered plant species in ex situ environments (cultivation in near nature-like replicas of the original site with as little human inference as possible over only certain periods of time, >30 years).     

Seed Bank Viability in Disturbed Longleaf Pine Sites

Some of the most species‐rich areas and highest concentrations of threatened and endangered species in the southeastern United States are found in wet savanna and flatwood longleaf pine (Pinus palustris Mill.) communities. Where intensive forestry practices have eliminated much of the natural understory of the longleaf ecosystem, the potential for reestablishment through a seed bank may present a valuable restoration opportunity. Longleaf pine sites converted to loblolly pine plantations and non‐disturbed longleaf sites on the Coastal Plain of North Carolina were examined for seed bank presence and diversity. Conducting vegetation surveys and examining the seed bank using the seedling emergence technique allowed for verification of the seed bank presence, as well as evaluation of the quality of the seed bank on disturbed longleaf pine sites. Forty‐three species and over 1,000 individuals germinated, and the seed banks of both the disturbed and non‐disturbed stand types contained species not noted in the vegetation survey. Although many of these species were considered weedy and typical of disturbance, numerous taxa were indicative of stable longleaf pine communities. This study confirms both the presence and quality of seed banks in highly disturbed former longleaf pine sites, suggesting that the seed bank may be an important tool in restoration efforts.     

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.     

Microsatellite primers for Texas wild rice (<Emphasis Type="Italic">Zizania texana</Emphasis>), and a preliminary test of the impact of cryogenic storage on allele frequency at these loci

Seed collections in gene banks are useful for the preservation of wild germplasm, providing inexpensive insurance for species that survive in conventional cold storage (–18 °C). Seeds that cannot survive these conditions must be pretreated with cryoprotectants and stored at liquid nitrogen temperatures, which presents unique technical and methodological challenges. Implicit in this approach is the assumption that these added manipulations do not change the genetic diversity of the preserved collections. We used polymorphic microsatellite markers for an endangered aquatic grass, Texas wild rice (Zizania texana), to conduct a preliminary evaluation of the effects of cryogenic preservation of mature embryos on genetic diversity. Using several statistical approaches, we show that allele frequencies did not change in collections of seeds that underwent cryopreservation (cryoprotected) compared to those samples that was not exposed to cryopreservation (control). The retention of the allelic diversity at the five loci examined suggests that there were no significant changes in genetic diversity due to treatments and that these protocols may be appropriate for ex situ conservation of genetically diverse wild germplasm.     

水松地理分布及其濒危原因

水松(Glyptostrobus pensilis)被列为国家一级保护植物,主要分布于我国的广东、广西、江西、湖南、福建、四川和云南等省区和越南。根据野外调查、查阅标本和文献,结合水松的化石分布资料,对分布区内水松的生长状况和分布区的自然环境概况进行了分析,讨论了温度、雨量、光照和土壤等生态因子对水松地理分布的影响。导致水松濒危的主要原因有：历史时期气候变迁导致水松分布区大幅度缩小和居群数量剧烈下降;水松对生境要求严格;人为破坏严重。根据水松濒危的原因和现状,建议加强对水松原产地的保护,特别是保护那些遗传多样性高的居群;重视水松居群周围生境的保护;以及采取人工繁殖和进行迁地保护等策略。     

Evaluating germinability of eight desert halophytes under long-term seed storage: Implications for conservation

Ex situ conservation in seed banks is a potential complementary conservation strategy for native plant species.It is well established that ex situ seed banking of native wild plants prolongs seed viability and thereby preserves genetic and species diversity for future use.We evaluated ex situ storage potential of eight halophytic species from deserts in the United Arab Emirates(UAE)by studying seed germination.Specifically,we examined the germinability of freshly collected seeds and seeds stored for three years in a seed bank.We also examined the effect of light conditions on fresh and stored seed germination.Fresh seeds of seven of the eight species tested had a higher germination rates under 12/12 h light/dark fluctuations than did those exposed to total darkness.Storage reduced light sensitivity in Halocnemum strobilaceum,Suaeda aegyptiaca,Salsola drummondii and Salsola imbricata,but increased the requirement for light in Arthrocnemum macrostachyum.In Anabasis setifera,storage decreased germination percentage when there was a 12-hour light/dark fluctuation,but increased germination rate when exposed to the dark treatment.Storage significantly reduced germination in both the light/dark and dark treatments in Suaeda vermiculata and S.aegyptiaca.Germination speed also responded differently to storage;whereas Timson's index significantly increased in A.macrostachyum and H.strobilaceum,it significantly decreased for S.drummondii,S.aegyptiaca and S.vermiculata.Germination of these species at a range of temperatures requires further testing;additionally,we strongly suggest that these laboratory findings be complemented by field studies.     

Temporal effects of agri-environment schemes on ditch bank plant species

Many of the agri-environment schemes (AES) implemented in the Western Peat District of the Netherlands have as their objective the conservation of the diversity of ditch bank plants. We investigated the effects of AES on ditch bank species in this area, using a dataset collected by 377 farmers who managed and monitored ditch banks during a 10-year period. We found that species richness has increased minimally over the last 10 years in ditch banks. Yet, we found no differences in increases in time between ditch banks with and without AES. In both ditch bank types plant species composition changed to species with higher nitrogen tolerance. Furthermore, species that disperse over long distances by water increased, whereas species with no capacity to disperse over long distances declined in both ditch bank types. This indicates that changes in vegetation composition in ditch banks are affected by other factors than AES.     

The study of genetic diversity patterns of Coffea commersoniana, an endangered coffee species from Madagascar: a model for conservation of other littoral forest species

Madagascar has 59 described species of Coffea, of which 42 are listed as critically endangered, endangered, or vulnerable by the criteria of the Red List Category system of the World Conservation Union (IUCN). The littoral forest of Madagascar is a distinctive type of humid evergreen forest restricted to unconsolidated sand located within a few kilometers of the Indian Ocean, now persisting only as small fragments with ca. 10 % of its original range remaining. In an attempt to understand the genetic diversity of Madagascan coffee species, we studied ex situ and in situ populations of Coffea commersoniana, an endemic species of the littoral forests of southeastern Madagascar and soon to be impacted by mining activities in that region. The in situ populations studied showed higher genetic diversity than the ex situ population. The genetic partitioning among the two in situ populations of C. commersoniana was high enough to necessitate keeping the two populations separate for restoration purposes. Based on these findings, recommendations for conservation management (in situ and ex situ) are made.     

Gaps in seed banking are compromising the GSPC’s Target 8 in a megadiverse country

Ex situ seed conservation is an effective strategy to help safeguarding plants from extinction. The updated Global Strategy for Plant Conservation’s (GSPC) Target 8 aims to include 75% of threatened plant species in ex situ collections by 2020, preferably in the country of origin. Halfway through the updated GSPC program, we evaluate the current state of knowledge and practice of ex situ seed conservation of threatened species from megadiverse Brazilian flora, contributing to this Target. We identify knowledge gaps and costs to achieve Target 8 through seed banking in Brazil within the time constraints of the GSPC and in a scenario of recent science budget funding cuts. Knowledge on seed storage behavior is available only for 24 Brazilian species (1.3%). Seed desiccation tolerance was concluded for 175 of 228 species, feasibly allowing safe storage of most Brazilian species at sub-zero temperatures. However, only 26 species (1.3%) are effectively banked in research institutions. Surprisingly, the percentage of banked threatened species hardly increased in the first 5 years since the update of the Target (0.55%, 2011–2015), and Brazil now faces the challenge of banking almost 1500 species during 2016–2020. Despite a major lack of commitment of Brazilian institutions and of knowledge to achieve the Target, the costs for banking the remaining species were estimated to be only US$3.9 million. We call for a nationwide coordinated effort of government agencies, policy makers and research institutions to include ex situ seed conservation in the environmental agenda to pursue achievement of the Target by 2020.