In situ or ex situ seed conservation: which is the more effective way to maintain seed longevity of an endangered cactus?

With restricted populations and a small number of individuals, Discocactus bahiensis Britton & Rose (Cactaceae) is an endangered species in Brazil and its capacity for the formation of seed banks in the soil and the maintenance of seed viability remains unknown. Thus, the aim of the present study was to determine the most efficient way to maintain viability during storage of seeds of D. bahiensis . Seeds were stored in paper bags and either kept in a cold chamber (7 ± 2°C) in the dark (ex situ conservation) or buried in the soil to a depth of 5 cm in an area of natural occurrence of the species (in situ conservation). Germinability of the seed banks was evaluated monthly for 20 months. During the first 10 months of storage, germinability of the seeds conserved in situ and ex situ was similar to that of recently collected seeds. After this period, a 70% reduction in germinability was found for the seeds maintained in situ and there was nearly complete loss of viability after 12 months of storage in the field (germinability < 10% in the last 8 months of the experiment), indicating the ability to form persistent soil seed banks. In contrast, the seeds stored in the cold chamber maintained greater than 70% germinability throughout the entire analysis period, demonstrating that ex situ conservation is the most efficient way to maintain the viability of the seeds of this endangered species.     

Comparison of genetic variation in populations of wild rice, Oryza rufipogon, plants and their soil seed banks

Wild rice, Oryza rufipogon, has endangered species conservation status and it is subject to in situ conservation in China. To understand the potential of the seed bank in species conservation and population restoration, this study compared the genetic diversity of O. rufipogon plants with that of its soil seed banks in two marshes. A total of 11 pairs of rice SSR primers were used and 9 were polymorphic. Allele frequencies of the seeds differed significantly from those of surface plants and varied between soil layers. Relatively more alleles and higher genetic diversity (H _e) were found in plant populations, relative to seed banks. The numbers of germinable seeds and the level of genetic variation in seed banks decreased with the increasing of soil depth, indicating a rapid seed loss. Genetic differentiation was detected between sites and between plant and seed populations, as well as among seeds of different soil strata. Rapid seed loss, partly dormancy loss, and nonrandom seed mortality are discussed as the possible contributors to the pattern of reduced genetic variation within seed banks, compared to plants. These could also be responsible for the considerable genetic differentiation between populations. The seed population held about 72% of the total genetic variation of O. rufipogon in each marsh, indicating the potential of seed banks for restoring population variabilities if the plant populations were lost.     

The contribution of the Millennium Seed Bank Project to <Emphasis Type="Italic">ex situ</Emphasis> plant conservation in Chile

For a long time in situ conservation has been the main approach used to protect Chilean plant diversity. However, due to the high level of endemism of its flora (50%) and an increasing human impact on wild areas, ex situ conservation has become an urgent requirement to avoid the extinction of plant populations and species. Since 2001, the Instituto de Investigaciones Agropecuarias (INIA), Chile, has been working in partnership with the Royal Botanic Gardens Kew, (Kew) through the Millennium Seed Bank Project (MSB) with the objective of conserving 20% of the Chilean flora as seeds in long-term storage. This seed conservation effort has focussed mainly on the endangered and endemic plants of the Chilean drylands. Towards the end of the first phase of the MSB some 1482 seed collections representing 850 species and subspecies have been collected and safely preserved in the INIA Seed Base Bank and duplicated at Kew. Almost 70% of the total species collected are endemic to Chile and several of them are endangered. Additionally, seed germination research has been conducted for nearly 400 species and seed collections have been used to propagate several threatened species. Germination protocols have been published and disseminated online. Over 4,500 herbarium vouchers have been collected, largely duplicated at Kew and at the national herbarium in Chile. As a result of the inputs of INIA and the MSB, collaboration has been extended to other national stakeholders, mainly for plant taxonomy and seed collecting. In this context two training courses have been run for 70 staff/students. This training has contributed to the raising of general awareness of the need for the long-term protection of Chilean plant diversity and to demonstrate the key role that ex situ seed conservation can play in meeting this need.     

A study on population genetic structure of Oryza meyeriana (Zoll. et Mor. ex Steud.) Baill. from Yunnan and its in situ conservation significance

In order to determine genetic diversity ofOryza meyeriana (Zoll. et Mor. ex Steud.) Baill., 12 enzyme systems encoded by 17 loci were electrophoretically analyzed in 164 individuals of seven populations from Simao Prefecture, Yunnan Province, China. In comparison with those seed plants with the same life history and breeding systems, as well as the other species in the genusOryza, the species shows rather low levels of genetic diversity (A = 1.1,P = 8.0 %, Ho = 0.004 and He = 0.015) within populations and high genetic differentiation among populations. F_st was up to 0. 649, suggesting that 64. 9% of total genetic variability exists among populations. Considering high genetic differentiation among populations from a limited geographic region, most of the populations of the species are worth being protected, and therefore, great natural protection regions should theoretically be established in which a great deal of populations should be involved for developingin situ conservation management. Meanwhile, some priory localities forin situ conservation ofO. meyerzana in Yunnan Province, were proposed. Project supported by the Grant of the President of the Chinese Academy of Sciences.     

<Emphasis Type="Italic">In vitro</Emphasis> and cryogenic preservation of plant biodiversity in Brazil

The Brazilian biomes (Amazon, Atlantic Forest, Cerrado, Caatinga, Pantanal, and Pampa) comprise one of the highest levels of plant diversity in the world; however, non-sustainable practices, deforestation, and land use have resulted in significant losses and fragmentation of the native forests. These ecosystems are now threatened and protection of their native plants through ex situ conservation is an urgent necessity. Cryopreservation and in vitro conservation are complementary options for securing and protecting Brazil’s native plant species because their potential economic value is critically important to develop strategies that will (1) support their sustainable utilization, (2) protect against the over-exploitation of species growing in natural habitats, and (3) conserve the genetic diversity of germplasm from species of different provenances. Biotechnological approaches will help to address future economic and environmental demands placed upon already at-risk species. Conserving seed germplasm ex situ provides an additional safeguard against the risks (e.g., loss due to disease, climate change) of field conservation. Moreover, seed banks and cryobanks permit the long-term conservation of a wider genetic base; this offsets the labor and space intensive costs of conserving in the active growing state. This paper is a compilation of the current status of strategies applied for conserving Brazilian native plant species.     

Ex Situ Conservation of Orchids in a Warming World

Whilst there is overwhelming scientific evidence that dramatic changes in regional climates are likely to occur throughout the 21st century, the scientific community remains uncertain how the effects of global heating will combine with other environmental factors to affect wild orchid populations. It is, however, likely that many populations will be affected adversely and that in situ conservation techniques by themselves will not be sufficient to prevent the extinction of many species. A range of complimentary ex situ strategies are discussed. Amongst these orchid seed banking has been shown to be an invaluable tool for conserving the maximum amount of genetic diversity in the minimum space and has the potential to enable the conservation of valuable material for possible re-introduction and habitat restoration programmes in the future. The Darwin Initiative project, ‘Orchid Seed Stores for Sustainable Use’ (OSSSU), is currently establishing a global network of orchid seed banks focussing initially on countries with high orchid biodiversity in Asia and Latin America. Particular reference is made to ex situ conservation in China, together with the urgent need to gather more data to determine which habitats and species are most at risk of extinction in the wild in the immediate future.     

Different plant provenance same seed tolerance to abiotic stress: implications for <Emphasis Type="Italic">ex situ</Emphasis> germplasm conservation of a widely distributed coastal dune grass (<Emphasis Type="Italic">Uniola paniculata</Emphasis> L.)

Preservation of genetic diversity within germplasm repositories represents an important tool for plant conservation. However, seeds must tolerate extreme levels of post-harvest desiccation and cold to realize benefits of ex situ storage. Factors including local climate and habitat impact expression of desiccation and freezing tolerance especially for widely distributed species. Our aim here was to understand the influence of a latitudinal gradient on seed desiccation and cryo-freezing tolerance. We sampled mature U. paniculata seeds from two geographically and genetically distinct populations then examined seed-water relations and germination following desiccation via equilibrium drying assays (0.5 to 91% RH; ?797 to ?12.9 MPa). Germination ability after drying and subsequent cryo-freezing treatments (?196?°C, 1 to 1440 min) was also evaluated. Seeds of both populations displayed similar reverse sigmoid moisture sorption isotherms characteristic of desiccation tolerant tissues. Furthermore, initial seed water potential (?63 and ?90 MPa) was considerably lower than the lethal limit (?20 MPa) identified for desiccation sensitive tissues. Final germination (range 58–93%) and temporal patterns differed significantly between populations following desiccation and cryo-freezing stress, but these germination responses were similar to initial germination. A higher proportion of non-germinated, yet viable seeds remained for the northern compared to southern population. Location does influence germination response, but differential germination is related to seed dormancy rather than desiccation or cryo-freezing sensitivity. Ex situ conservation of U. paniculata is therefore feasible across the latitudinal gradient studied here.     

Climate change and the African baobab (Adansonia digitata L.): the need for better conservation strategies

The baobab tree, with more than 300 uses and commercial value in EU and United States, has been identified as one of the most important trees to be conserved and domesticated in Africa. A decline in baobab populations because of changes in climate could have a negative effect on African livelihoods. Therefore, it is important to study the potential future distribution of this species and determine strategies for conservation. We used Maxent, 480 geo‐referenced records, present and future climatic and soil layers. Different general circulation models and scenarios were selected. Models were simulated for (i) All records, (ii) East Africa and (iii) West Africa species records. For each combination, the proportion of the present habitat that might remain suitable in the future was determined. These habitat proportions were compared with the Protected Areas in Africa. Although potential future distributions were different depending on model, scenario and records used, in all cases only a percentage of the present distribution was predicted to remain suitable in the future. Some countries were found to have no suitable habitat in the future. Recommendations for different conservation strategies include in situ conservation in Protected Areas; ex situ conservation in seed banks; and conservation through ‘sustainable utilization’.     

Genetic spatial clustering: significant implications for conservation of wild soybean (Glycine soja: Fabaceae)

Knowledge of spatial patterns of genetic variation within populations of wild relative species has significant implications with respect to sampling strategies for ex situ and in situ conservation. To study spatial genetic structure of wild soybean (Glycine soja Sieb. et Zucc.) at the fine scale, three natural populations in northern China were analyzed using inter-simple sequence repeat (ISSR) fingerprints for estimating kinship coefficients. A regression analysis of kinship coefficients against spatial distances revealed that individuals occurring close together tended to be more genetically related. The Sp statistic further indicated a comparable spatial pattern among the three wild soybean populations with similar Sp values (mean = 0.0734, varied from 0.0645 to 0.0943) detected across the three populations. Genetic patches were on average ca. 20 m in size, and the effective neighborhood sizes varied between 10 and 15 m. The spatial genetic structure evident in the wild soybean populations may be attributed to the restricted seed dispersal and predominant inbreeding mating system of this species. The detection of family structure in the populations of wild soybean has a significant implication for the effective conservation of the important genetic resources.     

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.     

Importance of <Emphasis Type="Italic">in vitro</Emphasis> technology to future conservation programmes worldwide

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail.     

Partnering conservation actions. <Emphasis Type="Italic">Inter situ</Emphasis> solutions to recover threatened species in South West Western Australia

South West Western Australia has a rich endemic flora of global significance. The threats facing this floral diversity are increasing in type, severity and scale, demonstrated by the rising numbers of species threatened with extinction. In particular, the root-rot pathogen Phytophthora cinnamomi is causing widespread destruction, threatening the survival of many of the region’s unique plants. In situ conservation of wild plants is considered the most essential component of a flora conservation program, but the ability to conserve some species adequately is often unachievable in the short term and urgent management intervention is required to prevent extinction. We present data on the status and management of wild populations of four threatened species from the region, including an ex situ program, and describe our efforts to bridge the gap between these two components. Such inter situ conservation recovery work enables monitoring of biological attributes, research into reproductive biology and collection of genetic material for further ex situ conservation, and provides the source of material for future restoration of wild populations.     

Genetic inference of epiphytic orchid colonization; it may only take one

Colonization of vacant habitat is a fundamental ecological process that affects the ability of species to persist and undergo range modifications in continually shifting landscapes. Thus, understanding factors that affect and limit colonization has important ecological and conservation implications. Epiphytic orchids are increasingly threatened by various factors, including anthropogenic habitat disturbance. As cleared areas (e.g. pastures) are recolonized by suitable host trees, the establishment and genetic composition of epiphytic orchid populations are likely a function of their colonization patterns. We used genetic analyses to infer the prevailing colonization pattern of the epiphytic orchid, Brassavola nodosa. Samples from three populations (i.e. individuals within a tree) from each of five pastures in the dry forest of Costa Rica were genotyped with neutral nuclear and chloroplast markers. Spatial autocorrelation and hierarchical genetic structure analyses were used to assess the relatedness of individuals within populations, among populations within pastures and among populations in different pastures. The results showed significant relatedness within populations (mean r = 0.166) and significant but lower relatedness among populations within a pasture (mean r = 0.058). Our data suggest that colonization of available habitats is by few individuals with subsequent population expansion resulting from in situ reproduction, and that individuals within a tree are not a random sample of the regional seed pool. Furthermore, populations within a pasture were likely colonized by seeds produced by founders of a neighbouring population within that pasture. These results have important ramifications for understanding conservation measures needed for this species and other epiphytic orchids.     

Interaction between ungulates and bruchid beetles and its effect on <Emphasis Type="Italic">Acacia</Emphasis> trees: modeling the costs and benefits of seed dispersal to plant demography

Integrative studies of plant–animal interactions that incorporate the multiple effects of interactions are important for discerning the importance of each factor within the population dynamics of a plant species. The low regeneration capacity of many Acacia species in arid savannas is a consequence of a combination of reduction in seed dispersal and high seed predation. Here we studied how ungulates (acting as both seed dispersers and herbivores) and bruchid beetles (post-dispersal seed predators) modulate the population dynamics of A. raddiana, a keystone species in the Middle East. We developed two simulation models of plant demography: the first included seed ingestion by ungulates and seed predation by bruchids, whereas the second model additionally incorporated herbivory by ungulates. We also included the interacting effects of seed removal and body mass, because larger ungulates destroy proportionally fewer seeds and enhance seed germination. Simulations showed that the negative effect of seed predation on acacia population size was compensated for by the positive effect of seed ingestion at 50 and 30% seed removal under scenarios with and without herbivory, respectively. Smaller ungulates (e.g., <35 kg) must necessarily remove tenfold more seeds than larger ungulates (e.g., >250 kg) to compensate for the negative effect of seed predation. Seedling proportion increased with seed removal in the model with herbivory. Managing and restoring acacia seed dispersers is key to conserving acacia populations, because low-to-medium seed removal could quickly restore their regeneration capacity.     

Criteria for assessing Italian <Emphasis Type="Italic">ex situ</Emphasis> collections of threatened plants

The ex situ conservation of plant species is a challenge for all signatories of the Convention on Biological Diversity to meet. In Italy, all conservation-related issues are supervised by the Ministry for the Environment and Protection of Land and Sea, but so far no national plan has been drafted, let alone funded, for ex situ plant conservation. As a contribution towards the establishment of a scientifically based policy, a national project has been launched, under the coordination of the Botanic Garden of Palermo. In its framework, the Botanic Garden of Pisa is developing a set of criteria for assessing Italian ex situ collections of threatened plant species. The criteria will be applied to datasets obtained by the Italian seedbanks through questionnaires. Requested data include resources and procedures, documentation systems, facilities and accessions. The expected results consist of the production of printed or electronic material concerning a) the technical-scientific profile of the Italian seed banks and of their collections; b) the ecogeographic profile of seed collections of selected species and c) the evaluation of the method used for gathering and analysing data. The project is currently under way and the results are due by September 2010. The documents and protocols which will be produced within the project will enable a stronger interaction of the Italian seed banks with other national and European seed bank networks.     

Patterns of seed longevity and dormancy in obligate seeding legumes of box‐ironbark forests,south‐eastern Australia

Current fuel loads and distribution suggest that fire events are infrequent and of a low intensity in the regenerated dry sclerophyll forests of the Victorian box‐ironbark ecosystem. However, many box‐ironbark species possess traits consistent with fire‐cued regeneration. It is unclear the degree to which human disturbance may have altered fire regimes in these forests. The infrequent and low‐intensity fire regime suggested by current fuel dynamics may pose a threat to the persistence of fire‐cued species. Obligate seeders such as those of the Fabaceae and Mimosaceae, common in box‐ironbark understoreys, may be particularly vulnerable if inter‐fire intervals exceed seed longevity. This study used seed burial trials to examine seed dormancy and longevity in five legume species to explore their capacity to regenerate under an infrequent, low‐intensity fire regime. All species displayed dormancy and longevity patterns consistent with other south‐east Australian legumes. Before burial, dormancy levels were high for all species (98–100%). After 3 years, storage under in situ and ex situ conditions, dormancy in Pultenaea prostrata remained at pre‐burial levels with virtually no seed becoming non‐dormant. Over time, some Acacia seed became non‐dormant under both in situ and ex situ storage, with the pattern varying among species. Longevity also varied between species. Variation in the dormancy and longevity patterns observed in these obligate seeder legumes suggests two strategies: (i) releasing a portion of soil‐stored seed from dormancy during the inter‐fire period to permit inter‐fire recruitment; and (ii) retaining most soil‐stored seed as dormant during the inter‐fire interval. Both strategies represent potential weaknesses under a long fire interval regime. The first relies on dormancy release translating to successful recruitment and requires ongoing inter‐fire input into the soil seed bank. The second relies on seed longevity exceeding the inter‐fire interval. Whether either is more suitable to coping with long‐term infrequent fire requires long‐term monitoring.     

Rapid genetic delineation of local provenance seed‐collection zones for effective rehabilitation of an urban bushland remnant

Abstract The rehabilitation of native plant communities in urban bushland remnants is an increasingly important activity requiring the collection of large amounts of seed. Best practice generally identifies that local seed are best, but how far does the local provenance extend? Using the DNA fingerprinting technique amplified fragment length polymorphism, we assessed genetic differentiation between potential seed source populations and the target population, Bold Park, a large and significant bushland remnant in Perth, Western Australia. For each of 15 species, analysis of molecular variance was used to partition genetic variation within and among populations. Genetic differentiation between Bold Park and potential seed source populations was assessed by non‐metric multidimensional scaling ordination, and statistically by Fisher’s exact tests. The partitioning of variation among populations (Φ_ST) varied from 0.66 for Santalum acuminatum to 0.04 for Mesomelaena pseudostygia. For eight of 15 species, Bold Park plants were completely or largely non‐overlapping with other populations in ordinations, suggesting genetic differentiation and a narrow provenance. Five species showed overlap between Bold Park and some other, but not all, populations sampled, with geographically closest populations generally undifferentiated. Only two species, Acanthocarpus preissii and Mesomeleana pseudostygia, showed little genetic differentiation between Bold Park and all other populations, suggesting a regional genetic provenance. These species can be classified into three broad provenance classes – narrow, local and regional – to help guide decisions about appropriate seed‐collection zones for the rehabilitation of urban bushland remnants.     

<Emphasis Type="Italic">In Vitro</Emphasis> Preservation of Spanish Biodiversity

Spanish territories contain many of the hot spots of plant biodiversity among European countries. Most of the Spanish territory is found in the Mediterranean basin and in the Canary Islands, a region of great floral singularity and diversity (Macaronesian bioregion). Therefore, an important effort must be made to contribute to its conservation. Several strategies can be considered, but seed conservation under standard conditions is the most resource-efficient method. However, the application of this methodology is not always possible for recalcitrant seeds or species for which vegetative propagation is necessary or convenient under some circumstances. Micropropagation is one of the measures suggested for preserving endangered species. During the 1990s, several in vitro culture protocols for Spanish endemics were established. The main purpose of this strategy was to obtain a considerable number of individuals to reduce the loss of natural populations. Likewise, diverse slow growth protocols were developed for this material. However, these efforts usually did not lead to the establishment of in vitro collections. The advantages and disadvantages of the in vitro conservation strategy will be reviewed for some cases. The establishment of the in vitro protocols together with the development of cryopreservation techniques created the ideal conditions to generate cryogenic collections. In this paper, we review the knowledge and experience accumulated during the last decades in micropropagation, slow growth, and cryopreservation for Spanish plant wild species. Their application in the development ex situ collections and their contribution toward an integrated system to conserve threatened species will be discussed.     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.