<Emphasis Type="Italic">In vitro</Emphasis> conservation of European bryophytes

The use of in vitro techniques for conservation has been rising steadily since their inclusion in The Convention on Biological Diversity and The Global Strategy for Plant Conservation. Unfortunately, bryophytes are often overlooked in conservation initiatives, but they are important in a number of large-scale ecosystem processes, i.e. nutrient, water and carbon cycling. There is a long history of the use of tissue culture in cultivating bryophytes, and many species respond well to in vitro techniques. For 6 yr (2000–2006), The Royal Botanic Gardens, Kew and the UK statutory conservation agencies supported a project for the ex situ conservation of bryophytes. Living and cryopreserved collections of UK threatened species were successfully established and the cryopreserved collection continues to be maintained. Other in vitro conservation collections are maintained over Europe, at botanic gardens, museums and by individual university researchers, but there is no coherent European collection of bryophytes for conservation, or standardisation of techniques. A major issue for many in vitro collections is the maintenance of within species genetic diversity. Such diversity is considered to be important, as it is the basis by which populations of species can adapt to new conditions and evolve. We are proposing to establish a European network for in vitro conservation of bryophytes. We envisage that this will include living collections, cryopreserved collections and spore collections. Conservation of genetic diversity would be a priority and the collections would provide a valuable resource for conservation initiatives and support research into rare and threatened species.     

Do living ex situ collections capture the genetic variation of wild populations? A molecular analysis of two relict tree species,Zelkova abelica and Zelkova carpinifolia

Botanic gardens and arboreta, particularly in regions where iconic relict trees naturally occur, play a vital role in the conservation of these species. Maintaining well-managed living ex situ collections of rare and threatened relict tree species provides an immediate insurance policy for the future species conservation. The aim of this research was to investigate the origin, representativeness and genetic diversity of relict trees kept in botanic gardens and arboreta. We used as a model two ecologically and biogeographically distinct members of the prominent relict genus Zelkova (Ulmaceae), which survived the last glaciation in disjunct and isolated refugial regions: Z. carpinifolia in Transcaucasia and Z. abelicea endemic to Crete (Greece) in the Mediterranean. Our study revealed substantial differences in the genetic diversity and the origin of living ex situ collections of the two investigated taxa. The living ex situ collections of Z. carpinifolia have relatively high representativeness compared with the global genetic variability of natural stands identified in a previous study. In contrast, Z. abelicea, which possesses an extraordinarily high genetic variability in natural populations, is clearly underrepresented in botanic garden collections. Moreover, all Z. abelicea trees investigated in this study most probably originated from a single region, the Levka Ori in western Crete. Thus, the ex situ conservation of Z. abelicea requires major planning and coordination efforts, including the establishment of well-documented collections in botanic gardens in Greece and especially on Crete. New living ex situ collections should be created using plant material collected from all of the mountain regions where Z. abelicea still occurs. Our study highlights the need for re-evaluating the existing living ex situ collections of trees and the development of new strategies for future conservation efforts in botanic gardens and arboreta. The coordination of conservation efforts between gardens must be enhanced to prioritize actions for the most threatened relict tree species.     

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.     

<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.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

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.     

The effectiveness of botanic garden collections in supporting plant conservation: a European case study

Europe has the greatest concentration of botanic gardens in the world, they cultivate extensive collections of plants that include samples of European threatened plant species. This study looks at the effectiveness of these collections in supporting species conservation. A three part study is presented: (1) the results of a survey and assessment of threatened plants in botanic gardens, as defined by the Bern Convention; (2) case studies illustrating current issues in the ex situ management of European threatened plant species; and (3) presentation of policy recommendations on further improving botanic garden contributions to European plant conservation. The survey indicated that of 119 European botanic gardens in 29 European countries, 105 are cultivating 308 of the 573 threatened plant species listed by the Bern Convention. The survey identified 25 botanic gardens in 14 countries undertaking 51 conservation projects focused on 27 Bern listed species. In particular this survey has established that the majority of taxa are held in a small number of collections, dominated by non-wild origin accessions, and are not adequately documented. The majority of specimens in botanic gardens are cultivated out of the range country and not contributing to a specific conservation project. We review the genetic representation and documentation of origin in collections. Existing plant collections contain representatives of populations, now lost in the wild and maintain samples of at least nine European plant taxa identified as 'Extinct in the Wild'. However, inadequate standards of record keeping has compromised the conservation value of many collections. We highlight the dangers of hybridisation and disease in ex situ collections. The results suggest that botanic garden collections are skewed towards horticulturally robust and ornamental species and do not fully reflect priorities as defined by the Bern Convention. Recognising the limitations of traditional botanic garden collections we propose that botanic gardens more effectively utilise their two core competencies, namely scientific horticulture and public display and interpretation. The unique horticultural skills resident in European botanic gardens could be more effectively utilised through the application of horticulture to the management of wild populations.Journal of the Royal Horticultural Society     

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.     

Conservation of important plants from the Ionian Islands at the Balkan Botanic Garden of Kroussia,N Greece: using GIS to link the in situ collection data with plant propagation and <Emphasis Type="Italic">ex situ</Emphasis> cultivation

The Balkan Botanic Garden of Kroussia (BBGK) is dedicated to the ex situ conservation of native plants of Greece and the Balkans. The BBGK has formulated a conservation strategy for the collection of wild plant material for propagation, prioritizing mainly the endemic, rare, endangered, threatened and vulnerable plants of Europe found in different regions of Greece. Its aim is to contribute to the implementation of Target 8 of the Global and European Strategies for Plant Conservation at local, regional and international scales. In order to (i) define the ecological profile of the in situ requirements preferred and/or tolerated by each selected species, (ii) develop rapid and effective species-specific propagation protocols, and (iii) improve the cultivation of species of conservation concern in BBGK’s nurseries and ex situ conservation sections, geographical coordinates and in situ collection data obtained for each taxon were imported into a Geographic Information System environment (GIS). This information was then linked with several digital GIS thematic layers, including topographic, geological, edaphic, climatic, precipitation and temperature data derived from digital databases. Based on this approach, sexual and asexual propagation of plants from the Ionian Islands were conducted and rapid and effective baseline protocols were developed for 29 taxa (species and subspecies); four are presented here in detail and species-specific ex situ propagation and cultivation guidelines are given. Most of the taxa originating from the Ionian Islands were propagated by cuttings (55.2%) or seeds (34.5%), while the rest were propagated by root division at a rate from 1.7 to 2. The first round of propagation achieved a success rate ranging from 15 to 50% for 3 taxa, from 60 to 80% for 8 taxa and from more than 80 to 100% for 16 taxa, while the ex situ cultivation of the wild and propagated plant material has, so far, been successful. The application of GIS exemplified here presents a sensible and invaluable tool with a broad-scale potential in enhancing the prospects of the ex situ conservation of priority species collected from diverse environmental conditions in man-made habitats such as botanic gardens.     

<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.     

<Emphasis Type="Italic">Lecomtedoxa plumosa</Emphasis> (Sapotaceae), a new tree species from Korup National Park,Cameroon

Summary   Lecomtedoxa plumosa Burgt (Sapotaceae), a new tree species from the southern part of Korup National Park in Cameroon, is described and illustrated. The flowers show the characteristics of the genus Lecomtedoxa: for example the staminodes are free and placed alternately to the stamens and corolla lobes. The leaves of the new species are clearly different from other Lecomtedoxa spp., but they look similar to the leaves of Gluema ivorensis, especially to those of the collections from Cameroon. In total 26 trees ≥ 10 cm dbh were found. The largest trees found were 36 m high and 74 cm dbh. The trees grow in primary rain forest, in clusters of up to 10 trees on 2 ha, mixed with many other tree species. The seed dispersal is ballistic. The conservation status of the species is assessed as Endangered, EN D.     

Phylogeny of <Emphasis Type="Italic">Litsea</Emphasis> and related genera (Laureae-Lauraceae) based on analysis of <Emphasis Type="Italic">rpb2</Emphasis> gene sequences

The relationship between Litsea and related genera is currently unclear. Previous molecular studies on these taxa using cpDNA and nrITS were unable to produce well-resolved phylogenetic trees. In this study, we explored the potential of the rpb2 gene as a source of molecular information to better resolve the phylogenetic analysis. Although rpb2 was believed to be a single-copy gene, our cloning results showed that most species examined possessed several copies of these sequences. However, the genetic distance among copies from any one species was low, and these copies always formed monophyletic groups in our molecular trees. Our phylogenetic analyses of rpb2 data resulted in better resolved tree topologies compared to those based on cpDNA or nrITS data. Our results show that monophyly of the genus Litsea is supported only for section Litsea. As a genus, Litsea was shown to be polyphyletic. The genera Actinodaphne and Neolitsea were resolved as monophyletic groups in all analyses. They were also shown to be sisters and closer to the genus Lindera than to the genus Litsea. Our results also revealed that the genus Lindera is not a monophyletic group.     

<Emphasis Type="Italic">Sporothrix inflata</Emphasis>, a root-inhabiting fungus of <Emphasis Type="Italic">Quercus robur</Emphasis> and <Emphasis Type="Italic">Q. petraea</Emphasis>

The anamorphic fungus Sporothrix inflata, known as a soil-borne fungus with worldwide distribution, was isolated for the first time from the cortex and central cylinder of living and dead roots of healthy and diseased oak trees (Quercus robur and Q. petraea). Isolation frequencies of S. inflata from oak roots varied according to the health status of trees, oak species, study sites, soil depth and root diameter. Colony morphology and growth rate of isolates are influenced by colony age and type of culture medium.     

<Emphasis Type="Italic">Ex situ</Emphasis> conservation programmes in European zoological gardens: Can we afford to lose them?

The role of ex situ activities for the conservation of biodiversity, and of zoos and aquaria in particular, is open to continuing debate. The present note highlights the conservation breeding potential of zoological gardens and aquaria in the European union, but it also recognises the lack of a convincing scientific and legal framework that encourages ex situ activities for ‘exotic’ species. If ex situ programmes are considered essential for global biodiversity conservation, the EU should not limit itself to regulating zoos through the zoo directive, but should actively promote and support their ex situ conservation activities.     

Molecular evolution of paclitaxel biosynthetic genes <Emphasis Type="Italic">TS</Emphasis> and <Emphasis Type="Italic">DBAT</Emphasis> of <Emphasis Type="Italic">Taxus</Emphasis> species

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.     

A revision of the genus <Emphasis Type="Italic">Podococcus</Emphasis> (<Emphasis Type="Italic">Arecaceae</Emphasis>)

Summary  A taxonomic revision of the palm genus Podococcus (Arecaceae) is presented. Two species are recognised: P. barteri, a species relatively widespread in a coastal band from Nigeria to the D. R. Congo and P. acaulis, a species previously considered conspecific to P. barteri, almost exclusively confined to Gabon. The taxonomic history, morphology, distribution and conservation status of the genus and each species are discussed