In situ conservation of crop wild relatives: a methodology for identifying priority genetic reserve sites

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1 An Integrated European In Situ Management Workplan: Implementing Genetic Reserve and On farm Concepts In situ conservation of crop wild relatives: a methodology for identifying priority genetic reserve sites Tested with case studies for Avena, Beta, Brassica and Prunus By S. Kell, N. Maxted, L. Frese, J.M. Iriondo, B. Ford Lloyd, K. Kristiansen, A. Katsiosis, C. Teeling and F. Branca

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3 AEGRO An Integrated European In Situ Management Workplan: Implementing Genetic Reserve and On farm Concepts In situ conservation of crop wild relatives: a methodology for identifying priority genetic reserve sites Tested with case studies for Avena, Beta, Brassica and Prunus By S. Kell, N. Maxted, L. Frese, J.M. Iriondo, B. Ford Lloyd, K. Kristiansen, A. Katsiosis, C. Teeling and F. Branca Front cover: Avena prostrata growing in southern Spain Photographer: Andreas Katsiotis Funded by the European Commission s Community programme on the conservation, characterization, collection and utilization of genetic resources in agriculture as a targeted action in accordance with Council Regulation (EC) no. 870/2004 Agreement no. 057 AGRI GEN RES Contract no. AGRI

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5 Contents 1.0 Introduction Background Approaches to in situ CWR conservation The genetic reserve concept Identifying priority sites for in situ CWR conservation: developing a common methodology Methodology for identifying CWR genetic reserve sites for a target crop gene pool Step 1: Taxon delineation Step 2: Selection of target taxa Step 3: Diversity analysis Step 4: Selection of target sites Application of the methodology to four crop case studies Crop case studies: overview and purpose Application of the methodology in the Avena case study Application of the methodology in the Beta case study Application of the methodology in the Brassica case study Application of the methodology in the Prunus case study Discussion Conclusion References... 36

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7 1.0 Introduction 1.1 Background Crop wild relatives (CWR) are species closely related to crops and are defined by their potential ability to contribute beneficial traits to crops (Maxted et al., 2006); they have been used in plant breeding since the early 20th century and have provided vital genetic diversity for example, to confer resistance to pests and diseases, improve tolerance to environmental conditions such as extreme temperatures, drought and flooding and to improve nutrition, flavour, colour, texture and handling qualities (Maxted and Kell, 2009). In monetary terms, CWR have contributed significantly to the agricultural and horticultural industries, and to the world economy (Maxted et al., 2008a; Maxted and Kell, 2009). Today, agricultural production is challenged by climate change (e.g., see Jones et al., 2003; Duveiller et al., 2007; Deryng et al., 2011; Li et al., 2011; Luck et al., 2011). The International Panel on Climate Change (IPCC, 2007) estimates that by 2100, crop yields will be reduced by as much as 40% in some regions unless climate change mitigation is undertaken. Breeders will therefore have to provide varieties able to cope with the impacts of changing growing conditions. Due to the breadth of genetic diversity inherent in CWR populations, which are adapted to a wide range of environmental conditions, they are likely to be needed more than ever before to maintain the adaptability of crops. Thus, CWR are a critical component of plant genetic resources for food and agriculture (PGRFA) and are vital for future food security; however, despite their recognized value, they have historically received relatively little systematic conservation attention. There are two primary techniques for CWR conservation: in situ, primarily in natural habitats managed as genetic reserves (GRs) (see section 1.3) and ex situ as seed in gene banks. Historically, CWR conservation has focused almost entirely on ex situ collection and storage, but it can be argued that in situ conservation is more appropriate because the genetic diversity inherent in and between wild CWR populations is constantly changing in response to their environment; therefore, CWR populations are a component of natural ecosystems that cannot effectively just be maintained ex situ (Maxted et al., 2008a). A number of recent initiatives have raised the profile of CWR and put them on the international conservation agenda. However, conservationists and policy makers are faced with the difficult challenge of how to conserve the large number of CWR species and the genetic diversity that they contain. If a broad definition of a CWR is used (i.e., all the species in the same genus as a cultivated plant), there are more than 16,000 crop and CWR species in the territories of the EU Member States 13,875 of these are native and at least 2665 are endemic (Kell et al., 2008). CWR are under threat from habitat loss, agricultural intensification, over collection, climate change and lack of conservation attention, yet only 9% of PGR accessions in European gene bank collections are CWR (Dias et al., 2011), while most wild populations in situ are not actively monitored and managed in or outside protected areas (PAs) (Maxted et al., 2008b). There is therefore a real challenge to the nature conservation and PGRFA sectors to conserve these valuable resources. The AEGRO project was initiated to fill some knowledge gaps and to provide a range of practical tools for in situ CWR conservation as a contribution to an integrated EU PGRFA conservation strategy. This report presents a methodology for the development of a CWR in situ conservation strategy for target crop gene pools that can be applied both within Europe and elsewhere in the world. The methodology has been tested and applied in four crop case 3

8 studies (Avena, Beta, Brassica and Prunus) by partners in the AEGRO project and this report presents an analysis of the application of the methodology to each case study with the aim of illustrating how such a generic methodology can be successfully applied across a range of different crop gene pools. 1.2 Approaches to in situ CWR conservation There are various approaches to achieving the systematic conservation of CWR diversity; however, two distinct but complementary approaches may be characterized as floristic and monographic (Maxted et al., 2011). These approaches relate to the breadth of coverage of the CWR conservation strategy a floristic approach involves the development of a CWR conservation strategy for CWR diversity that occurs in a defined geographical area (which may be a sub national area such as an administrative unit or protected area, a whole country, a supra national region, or even the whole world), while a monographic approach is restricted to certain crop gene pools, but like the floristic approach may be carried out at any geographical scale (Maxted et al., 2011). The floristic approach is comprehensive because it attempts to encompass all CWR diversity that occurs within a geographical unit; however, while being comprehensive for the geographical unit, the full geographic range of an individual taxon may or may not be included, depending on whether it is endemic to that geographical unit. The monographic approach focuses on CWR diversity within target crop gene pools which are usually identified on the basis of their perceived value for food security and/or economic stability. Both approaches will ultimately conclude with the systematic conservation of priority CWR diversity via a network of conservation sites and genetic reserves, with backup in ex situ collections (Maxted et al., 2011). Whether a floristic or monographic approach is taken is likely to depend on: a) the quantity and quality of existing data and b) the resources available to prepare the conservation strategy. The scope of the parent organization undertaking the conservation may also impact the approach; for example, an international cereal research institute is likely to focus monographically on cereal crops, while a national biodiversity institute is likely to adopt a more floristic approach. It is worth noting that if the goal is to maximize CWR diversity, it is likely that both approaches need to be combined (Maxted et al., 2011). In Europe, a combined floristic and monographic approach is being promoted and taken forward in the context of the EU funded project, PGR Secure (www.pgrsecure.org). The development of national CWR conservation strategies (taking the floristic approach) and a European strategy (taking the monographic approach for high priority crop gene pools) will contribute towards an overall European strategy for PGRFA conservation. 1.3 The genetic reserve concept A genetic reserve is defined as the location, management and monitoring of genetic diversity in natural populations within defined areas designated for long term conservation (Maxted et al., 1997a). The concept combines in situ conservation with active management and a longterm approach. The rationale for this type of conservation is that it is a) applicable to all plant species, b) allows for continued evolution and c) allows for multiple taxon conservation. Moreover, it conserves the genetic diversity of the target taxon in a dynamic way, as well as its habitat and all existing biotic and abiotic interactions (including humans). 4

9 Several approaches to GR conservation can be identified, each with different aims and strategies, depending on the approach (see Maxted and Kell, 2009). For example, the aims for CWR GRs in Europe are to conserve genetic diversity in the widest range of priority CWR taxa at the European scale; therefore, the aim is to design a network of reserves that adequately and efficiently maintains the genetic diversity of the target taxa. When we talk about adequately maintaining the genetic diversity of target taxa, we mean conserving a good representation of the genetic diversity of adaptive and agricultural value present in such taxa. Similarly, by efficiently we mean to obtain this goal using the minimum number of GRs. Coordination with ex situ holdings and crop databases is an important part of the genetic reserve concept (Maxted et al., 1997a). Ex situ seed banks can be a relevant component in the functioning of GRs as they provide a back up of genetic diversity in case any catastrophe should occur. Furthermore, they facilitate information exchange, access for breeding and other research, and promote use. The end point of the methodology described and discussed in this report is the identification of ideal GR sites. However, it is useful to briefly review the steps that follow, once the location of a GR has been decided. Firstly, it is necessary to consider the design of the reserve. Some of the factors to take into account are a) the shape and size of the reserve, b) target taxon population size, c) political and socio economical factors and d) the possibility of establishing networks and corridors with other neighbouring reserves. Secondly, a genetic reserve management plan needs to be developed. A management plan is a planning tool that contains a set of prescriptions and interventions to meet the objectives of the GR (Heywood and Dulloo, 2005). The minimum elements of a management plan have been described in detail by Maxted et al. (2008c). Essentially, a management plan is composed of three basic elements: i) assessment of the taxon, population and site, ii) establishment of management targets, and iii) prescription of conservation actions. Evaluation is an essential component for management success. The purpose is to identify and determine the factors that affect population viability and the maintenance of genetic diversity in the populations. The establishment of clear, precise management targets is needed to obtain and evaluate the desired results and to measure success (Elzinga et al., 1998). Management and monitoring approaches depend on the definition of specific targets (e.g., what status do we want to achieve in the population, or under what circumstances are we going to take a particular action?) Some examples of management targets can be to keep population size over a certain number of individuals, not to allow a decline in population size over a certain percentage in one year, or to keep a specific allele that is key for pathogen tolerance in a gene at a frequency above a certain percentage. Finally, management prescription includes all types of actions that can be executed on the target population, its habitat and the surrounding community to maintain ecological conditions and processes that are compatible with and necessary for the survival and maintenance of the genetic diversity in the target population (Maxted et al., 2008c). Management prescription also includes monitoring, which is a procedure to assess the effects of the management actions that are implemented. Since as a result of monitoring we may be able to conclude that our management actions are not producing the results we wanted, management prescription must also consider what actions to take when things do not go the way they are supposed to. 5

10 1.4 Identifying priority sites for in situ CWR conservation: developing a common methodology The establishment of GRs for CWR is a priority in order to maintain a broad range of genetic diversity within and between populations; however, with a large number of species to conserve, a systematic approach to the identification of GR sites is needed to maximize resource use. In this report, we present a generic methodology that can be used to prioritize taxa on the basis of their potential use for crop improvement and relative threat status, gather the necessary data to undertake diversity and gap analysis for target taxa, and select the most appropriate CWR GR sites. The methodology is built on those proposed by Maxted et al. (in prep.), Maxted et al. (2008d) and Maxted and Kell (2009), which address floristic and monographic approaches to CWR conservation. Although it may be necessary to adjust parts of the methodology according to the specific biological, ecological and geographical attributes of individual crop complexes, it provides a generic framework for the conservation of any crop gene pool. There are four basic steps in the methodology: a) taxon delineation, b) selection of target taxa, c) diversity analysis and d) selection of target sites. Within each of the four steps, precise instructions are provided on how to develop the strategy. The end point of the methodology is the identification of ideal CWR GR sites. The political and legal steps that need to be taken beyond this point to establish the GRs are not part of the methodology. The next step beyond the methodology for identification of GRs is to make recommendations for site and population management (see Maxted et al., 2008c). The methodology was first made available to AEGRO project partners via the project intranet as part of the AEGRO Helpdesk. After testing and further development, the methodology was made available in the public domain (see as part of the CWR In Situ Strategy Helpdesk, which is provided as a guide and information facility for national programmes, research institutes, NGOs, protected area managers or individuals involved in the development of a CWR in situ conservation strategy. The methodology is presented in the next section. For more detailed step by step guidance on implementing the methodology, including a list of data sources, the reader is referred to the Helpdesk. 6

11 2.0 Methodology for identifying CWR genetic reserve sites for a target crop gene pool 2.1 Step 1: Taxon delineation Background The starting point for a crop gene pool CWR conservation strategy is a list of target taxa; therefore, for the target crop gene pool it is necessary to: 1. Generate a list of taxa that occur in the crop gene pool. Although not all the taxa in the gene pool will necessarily be immediately included in the CWR conservation strategy, the complete list of taxa provides a reference point for future potential conservation actions of lower priority taxa. 2. Generate a list of taxa that occur within the defined geographic range of the conservation strategy (i.e., national, regional or global). These may be both native and introduced, but the conservation strategy is most likely to focus on native species. To achieve these two steps, online information sources and/or literature (monographs, cropspecific studies etc.) need to be consulted (see Maxted and Guarino, 2003). At this stage, it is necessary to adopt an accepted taxonomy to form the basis of the taxon list and the subsequent conservation strategy. The list of taxa should show the accepted taxon name and authority and list primary synonyms with authorities. This is important because different information systems use different accepted taxonomies; therefore, when searching for information on a specific taxon it could be possible to miss important information if synonymy is not taken into account. Step 1 actions list the taxa in the crop gene pool 1. Produce a list of all taxa within the crop gene pool, including both accepted names and synonyms with authorities. To achieve this, consult available online information sources and/or consult the literature (monographs, crop specific studies etc.). 2. If applicable, list the taxa that occur within the geographic range (i.e., national or regional) of the CWR conservation strategy. Several online data sources can be consulted to provide information on the distribution of the taxa. However, good starting points are the Crop Wild Relative Catalogue for Europe and the Mediterranean, accessible via the Crop Wild Relative Information System (CWRIS and the Global Biodiversity Information Facility (GBIF For national level data, the same data sources can be consulted, as well as national Floras and databases, as applicable. To access these online data sources, further information on other sources of national level data, and for advice on how to use CWRIS, GBIF and other online information sources, visit the online Helpdesk data sources web page at 7

12 2.2 Step 2: Selection of target taxa Background In general, it is not practical to attempt to actively conserve all the taxa within the crop gene pool due to resource limitations; therefore, we need to prioritize and select taxa from the list that will be proposed for active conservation. Factors that can be used to ascribe value and establish conservation priorities include (Maxted et al., 1997a): 8 Current conservation status Socio economic use Threat of genetic erosion Genetic distinctiveness Ecogeographic distribution Biological importance Cultural importance Cost, feasibility and sustainability Legislation Ethical and aesthetic considerations Priorities of the conservation agency For CWR, an initial, simple prioritization on the basis of socio economic use of the associated crop (a step which will already have been taken in selecting the target crop gene pool) and relative threat has been proposed (e.g., Ford Lloyd et al., 2008; Magos Brehm et al., 2008). In addition, Maxted and Kell (2009) proposed that within each crop gene pool, the closest wild relatives should be afforded higher conservation priority over the more distantly related species because these are the taxa that can more easily be used in crop improvement using conventional breeding methods. However, the literature on the taxa within the target crop gene pool should be thoroughly searched to check for cases where a more distantly related taxon has been highlighted as a gene donor (or potential gene donor), and these taxa should also be afforded conservation priority. Of these prioritized taxa, those in most urgent need of conservation action (i.e., those with a very limited geographic range often rare or endemic taxa and/or known to be under threat), are given precedence. This methodology therefore primarily targets the taxa that are most closely related to the crop species (or that have shown promise in crop improvement programs) and that are threatened or have restricted distribution ranges. However, ideally, national and regional in situ networks of CWR GRs should in the long term be expanded to ensure that all taxa of potential importance for crop improvement are actively conserved. In particular, selected populations of the primary and secondary wild relatives that are widespread and common should be actively conserved throughout their range, ensuring that populations representing the extremes of the range (both geographically and topographically) are conserved. Individual populations of these taxa may harbour important genes adapted to particular environmental conditions genes that may confer important traits to improve crops in the future. Populations of these taxa that already occur within PAs should also be monitored. In many

13 cases, if a floristic approach is taken, it is possible to establish a reserve that conserves multiple CWR taxa, which, when possible, has obvious advantages. There are two stages to the selection of target taxa: a) creation of a level 1 prioritized list based on actual or potential use as gene donors, and b) creation of a level 2 prioritized list based on threat and/or distribution. In this methodology, the two steps are presented sequentially (i.e., the level 2 prioritized list is based on the level 1 prioritized list). The advantage of this approach is that in cases where there is limited information on the distribution of the taxa and/or for gene pools containing a very large number of taxa, the level 1 prioritization narrows the list of taxa down to those that are likely to be most important as gene donors for crop improvement and further information is only sought for that list of taxa. The disadvantage of this approach is that some of the more distantly related taxa in the gene pool that are threatened or have restricted distributions may be missed in the conservation planning process. Therefore, in cases where a gene pool contains a relatively small number of taxa or where distribution data are readily available for all the taxa, it is desirable to undertake the prioritization in the reverse order by collating threat and distribution data on all taxa in the gene pool first, then applying the second level of prioritization based on potential use as gene donors. Using this approach, more distantly related taxa that are threatened or have restricted distributions can be highlighted as a conservation priority on that one criterion, and even though they may still not be given the highest level of priority for immediate conservation action, they may be promoted as candidates for conservation at a later date. Furthermore, if it is not immediately possible to put in place in situ conservation measures for these taxa, they can be earmarked for collection and storage in ex situ collections. Step 2 actions a) create a level 1 prioritized taxon list At this stage, the aim is to prioritize the taxon list on the basis of value or potential value as gene donors for crop improvement, as follows: 1. Organize the list of all taxa within the crop gene pool according to their degree of relationship to the crop. To achieve this, search the available literature on the crop complex. Taxa should be organized into a table showing primary, secondary or tertiary wild relatives using one of the following three methods: a) Where genetic information is available and taxa have been classified using the Gene Pool concept (Harlan and de Wet, 1971), organize the taxa into the table listing those in GP1B as primary wild relatives, those in GP2 as secondary wild relatives and those in GP3 as tertiary wild relatives. The Gene Pool concept works like this: GP1A: cultivated forms of the crop GP1B: wild or weedy forms of the crop GP2: the coenospecies (less closely related species) from which gene transfer to the crop is possible but difficult using conventional breeding techniques GP3: the species from which gene transfer to the crop is impossible, or if possible, requires sophisticated techniques, such as embryo rescue, somatic fusion or genetic engineering. b) Where genetic information is not available, if possible, substitute the Gene Pool concept with the Taxon Group concept (Maxted et al., 2006), which provides a proxy for taxon genetic relatedness. Organize the taxa into the table listing those in TG1b as primary wild 9

14 relatives, those in TG2 as secondary wild relatives, and those in TG3 and TG4 as tertiary wild relatives. The Taxon Group concept works like this: TG1a: crop TG1b: same species as crop TG2: same series or section as crop TG3: same subgenus as crop TG4: same genus. c) For crop genera that have not been classified using the Gene Pool concept and not subclassified into sections and subgenera, the available information on genetic and/or taxonomic distance must be analysed to make reasoned assumptions about the most closely related taxa. Whichever system is used, ensure that references are provided to substantiate the assumptions made about taxon relatedness. 2. Select the taxa from the categorized list that occur within the geographical area of the CWR conservation strategy by matching against the geographically defined list of taxa created under Step 1. You will now have two tables, both categorized according to their degree of relationship to the crop one showing all taxa in the crop genus, the other showing the taxa that occur within the geographical area defined by the CWR conservation strategy. 3. In both tables, flag the taxa that have been identified as gene donors or potential gene donors. To achieve this, consult online information sources, such as GRIN Taxonomy and/or carry out literature searches using online library databases. Specialist libraries can also be consulted, such as those housed in gene banks, botanic gardens and other research institutes. Consult the online Helpdesk data sources page for further information (http://aegro.jki.bund.de/aegro/index.php?id=193). Provide references for any taxa flagged as gene donors or potential gene donors in the table. These may be in the tertiary list, as well and the primary and secondary lists. 4. Make a separate list of the primary and secondary wild relatives and any tertiary wild relatives that have been identified as gene donors or potential gene donors from the list of taxa within the geographical area defined by the CWR conservation strategy. This list (level 1 priority list) now forms the basis for the second level of prioritization under Step 2b. Step 2 actions b) create a level 2 prioritized taxon list At this stage, the aim is to prioritize the level 1 priority taxon list on the basis of threat status. However, as already noted, this level of prioritization can be applied to all taxa listed under Step 1, followed by prioritization on the basis of actual or potential value for crop improvement, depending on the size of the gene pool and on the availability of distribution and/or threat data. Therefore, it is also possible to carry out the prioritization process in reverse order (i.e., by carrying out Step 2b followed by Step 2a). There are two steps to this selection process: 10

15 1. Select the taxa in the level 1 priority list (or Step 1 list) that are known to be under threat. Where existing evidence is available to show that a taxon in the list is under threat, these taxa should automatically be prioritized for conservation action. To find out whether a taxon is known to be under threat: Search the IUCN Red List of Threatened Species to find out if any of the taxa are listed. Where possible, consult national or regional Red Lists to see whether a taxon in the list is considered threatened at national or regional level. If a taxon is not included in the IUCN Red List, or in a regional or national Red List (i.e., it has not been formally assessed using a set of Red List criteria), it does not mean that it is not threatened. A literature search may reveal important information about the threatened status of a taxon. To achieve this, carry out literature searches using online library databases. Specialist libraries can also be consulted, such as those housed in gene banks, botanic gardens and other research institutes. Consult the online Helpdesk data sources page (http://aegro.jki.bund.de/aegro/index.php?id=193) for further information and advice on how to carry out these searches. 2. Select the taxa in the level 1 priority list (or Step 1 list) that are not known to be under immediate threat, but are known to have limited distribution ranges. At this stage, a degree of objectivity is required, since there is no clear dividing line between a taxon with a limited distribution range and one with a distribution range that is deemed to enable classification of the taxon as one not in immediate need of conservation action, unless very detailed information is already available about the taxa. However, where the range of a taxon is known, the methodology proposed by Ford Lloyd et al. (2008, 2009) can be used as a guide when establishing taxon conservation priorities at regional level (e.g., across Europe). Generally speaking, taxa that are known to be endemic to a country or subnational unit or those that occur in only a few countries or subnational units are more likely to be under threat at regional level. Similarly, at national or subnational level, available information must be gathered on the range of the taxa in order to establish which are most likely to be threatened by their limited distribution range. To find the distribution range of the taxa: At European level, the Crop Wild Relative Catalogue for Europe and the Mediterranean (Kell et al., 2005) which can be accessed via the Crop Wild Relative Information System (CWRIS provides a list of country or subnational occurrences for taxa throughout Europe. At national or subnational level, a good starting point is GBIF, which provides known taxon occurrences through data provided by organizations managing biodiversity data around the world (e.g., ex situ germplasm collections and herbarium data). For a finer level of detail, search available national databases and literature sources. Taxon specialists, conservation agencies and protected area managers are further important sources of information. Consult the online Helpdesk data sources page (http://aegro.jki.bund.de/aegro/index.php?id=193) for further information and advice on how to carry out these searches. 11

16 After carrying out Step 2, you now have a reduced list of taxa that have been selected on the basis of their value as gene donors and relative threat. This list of target taxa now forms the basis for immediate conservation planning for the crop gene pool. 2.3 Step 3: Diversity analysis Background Once the priority list of CWR species has been identified (Step 2), the next step is to collate the available ecogeographic information to assist in further formulation of the CWR conservation strategy 1. This involves the collation and analysis of geographic, ecological, environmental and genetic data. These data are predictive and aid the location of the CWR taxonomic (inter taxa) and genetic (intra taxon) diversity that can then be targeted for conservation. As the goal is to maximize conserved genetic diversity, information on the partitioning of genetic diversity across the ecogeographic distributions of the target taxa is useful for identifying sites or combinations of sites of maximum diversity. However, even with rapidly decreasing costs of analysing genetic diversity, this information may be extremely limited; in which case, analysis of ecological and environmental data associated with the sites at which the populations occur can be used as a proxy for genetic diversity. The culmination of the diversity analysis should be a set of areas with high concentrations of the priority CWR species and populations of CWR taxa containing or thought to contain complementary and/or unique genetic diversity. Step 3 actions 2 a) carry out an ecogeographic diversity analysis At this stage, the aim is to collate all available ecogeographic data associated with the target taxa. Assuming the taxonomic data has already been collated in Step 1, the remaining data are of three main types: Geographic Genetic Ecological/environmental 1. Gather geographic (distribution) data on the target taxa. Data are of two types coordinate and descriptive. Ideally, coordinate data should be used for accuracy (however, even coordinate data can sometimes be misleading, depending on the accuracy and quality of the original data). Descriptive data can be converted to coordinate data by consulting gazetteers. A good starting point for the collation of geographic data is GBIF. Data downloaded from GBIF can then be supplemented by consulting a range of other data sources, such as EUNIS (for Europe) and national or specialist taxonomic databases. Consult the data sources page in the Helpdesk for further information on collating distribution data. At this stage in the analysis, issues of data quality have to be taken into account and steps may need to be taken to improve the accuracy of the distribution data to remove any erroneous entries. For example, a criterion used in the AEGRO project is that only population occurrences with geographic coordinates that have two decimal digits 1 Note that if distribution data are readily available for the entire gene pool (i.e., not just the target taxa identified under step 2), diversity analysis can be undertaken for all taxa. 2 Although the actions are presented sequentially, it is not necessary to carry out ecogeographic diversity analysis before undertaking a complementarity analysis. 12

17 or more are used in the analysis. Another limitation is that the availability of occurrence data may be very heterogeneous across the range of the target taxon this needs to be taken into account when making decisions on the selection of target sites (Step 4). Where distribution data are too sketchy or otherwise incomplete or inaccurate, it may be necessary to recommend that a detailed ecogeographic survey is undertaken before further analysis. 2. Gather genetic data on the target taxa. Genetic diversity analysis is only possible where the necessary information already exists or where resources permit the generation of novel genetic diversity information. There are two types of genetic diversity information of interest for the establishment of genetic reserves and for backup in ex situ collections: intra population and inter population diversity. The precise method of generating genetic diversity information is taxon specific. Decisions regarding the type of genetic analysis to undertake can be based on existing studies of related taxa or taxa sharing similar biological attributes. Literature searches can be undertaken to obtain this information, as well as consulting specialist databases and taxon experts. 3. Gather ecological and environmental data on the target taxa. Ecological and environmental data associated with the target taxa can be of two types: actual (i.e., data directly linked to a taxon) or secondary (i.e., data indirectly linked to a taxon via the attributes of the site in which it is found). Actual ecological and environmental data can be sourced by obtaining characterization and evaluation data associated with ex situ accessions, and/or by consulting the available literature on the target taxon for example, there may be published or grey literature as a result of ecological studies of the taxon or of associated taxa that occur in the same habitats or by collecting fresh data in the field. Secondary data are obtained by gathering data associated with known locations of a taxon (e.g., climate, soil type, geological substrate, habitat type, altitudinal range and land use). Some of these data are readily available in the form of Geographical Information System (GIS) files, which are overlaid with the distribution data, and from which inferences can be made about the ecological preferences of a taxon. Consult the data sources page for further information on gathering ecological and environmental data. 4. Analyse the ecogeographic data. The data collated is now analysed to build detailed taxon ecogeographic profiles. GIS programs such as ArcGIS or DIVA GIS can be used to create distribution maps overlaid with ecological, environmental and genetic data, and locate complementary genetic reserve locations. The analysis may be simple to complex, depending on availability of data, expertise, time and resources. The data should also be imported into an appropriate information management system from which standard taxon data sheets can be extracted to form the basis of genetic reserve proposals and management plans. Step 3 actions b) carry out a complementarity analysis Complementarity analysis may also be undertaken. This aims to maximize taxonomic diversity conservation in the minimum number of sites and may be useful when dealing with gene pools containing a large number of taxa or for multiple gene pools. The GIS program, DIVA GIS (see Hijmans et al., 2001) is useful for undertaking complementarity analysis and is available for download free of charge. 13

18 2.4 Step 4: Selection of target sites Background In some cases, the range of the target taxon will define the precise site or sites where active in situ conservation is needed. Obviously, a taxon that is known only to occur at one location and is considered a high priority as a potential gene donor, then that single location must be targeted for reserve establishment. Where the geographic range of the target taxon is broader, sites should be selected that represent the widest range of ecogeographic characteristics as possible. Once the target taxon distribution has been identified and mapped, and diversity analysis undertaken (Step 3), protected area (PA) overlays are used to ascertain whether the target taxon populations occur within the boundaries of an existing PA. CWR, like any other group of wild plant species, are located both within and outside existing PAs; however, the most efficient approach in the first instance (to avoid purchase of sites to establish genetic reserves) is to establish CWR genetic reserves within existing PAs (Maxted et al., 2007). Therefore, the most appropriate PAs (e.g., national parks and heritage sites) within which to locate genetic reserves should be identified. GIS analysis using PA shape files provides an indication of which PAs contain populations of the target taxa. In addition, this method can be used to predict which PAs contain high concentrations of CWR diversity. To be certain that the populations do exist within the PA(s), it is necessary to confirm their presence before genetic reserve establishment is recommended. This information is not always easy to obtain; however, it may be possible to contact the agency responsible for the management of the PA to see if they have an inventory of taxa available at the site or whether it is possible for site staff to confirm the presence of the taxon. If possible, ground truthing by visiting the site(s) personally should be undertaken. This is of course subject to available time and resources. Where target taxon populations are found to already occur within existing PAs, these populations should be prioritized for inclusion in the CWR genetic reserve network on the basis that they have already been afforded some degree of protection, even if only by default. However, it is important to stress that even though a target taxon population may occur within the boundaries of a PA, this does not automatically mean that the population is actively conserved. On the contrary, few PAs are established to conserve specific target taxa, and those that have often tend to focus on animal conservation. To conserve the range of genetic diversity inherent in CWR populations, active site management and monitoring is needed (see Iriondo et al., 2011) some PAs do not even have management plans, and those that do are often limited by financial resources and lack of capacity to put the plan into practice. In cases where a few to several PAs are found to contain populations of a target taxon, results of the diversity analysis can be used to select sites that best represent the ecogeographic diversity within the target taxon. A further consideration for the selection of PAs is the option for multiple taxa genetic reserves. Analysis of all target taxa within the crop gene pool (and preferably across several crop gene pools) may reveal that some PAs contain populations of more than one taxon. In terms of expediency of resource use, multi taxa reserves have obvious advantages over those that only contain a population of one taxon. Where target taxon populations do not already occur within existing PAs, these populations should also be prioritized for inclusion in the CWR genetic reserve network on the basis that 14

19 they have not already been afforded any degree of protection; especially for rare or threatened species. Obviously, justifying the need for and actually establishing new PAs will involve a significant initial injection of time and resources. Nomination of genetic reserves at the target locations may of course be hindered by a range of socio political factors, such as legal issues, land use conflicts, issues of land ownership, or lack of local support. Therefore, if possible a range of alternative sites should be recommended and ranked according to their suitability based on ecogeographic considerations. Step 4 actions select and prioritize target sites This is the final stage in the process, leading to the identification of CWR genetic reserves, which should ideally be assigned a priority ranking so that if for some reason a genetic reserve cannot be established at the best site (e.g., due to land use conflict or legal complications), the next best site is promoted, and so on. 1. Ascertain whether one or more populations of the target taxa occur within the boundaries of existing PAs. To achieve this, overlay target taxon distribution data with protected area shape files. A GIS program such as ArcGIS can be used for this purpose. PA data are available free of charge for use for scientific research, which are imported into the GIS program, along with the coordinate data collated under Step If one or more populations of a target taxon appear to fall within the boundaries of one or more existing PAs, follow up the analysis by verifying the presence of the taxon at the site. This is not always easy or possible, but it is important to try. If it is not possible to visit the site personally, contact the agency responsible for the management of the PA and ask if they have a recent taxon inventory available. If no recent inventory is available, ask if site staff are available to verify the presence of the taxon. If verification is undertaken by a third party it will be necessary to arrange for voucher specimens or at least photographs of the plants to be sent to ensure the correct identification has been made. 2. Select sites for the establishment of CWR genetic reserves. At this stage, there are a number of different variables to take into consideration, depending on the range of the target taxon and other factors: For taxa that occur in only one location, it is obvious that this location must be recommended for genetic reserve establishment, regardless of whether the population exists within or outside an existing PA. For taxa that occur in more than one location, the results of the diversity analysis carried out under Step 3 need to be added into the equation. There is no one fixed way of decision making at this stage, but first and foremost, since the aim is to conserve the maximum genetic diversity within and between populations of the target taxa as possible, then sites that are most likely to represent that diversity should be selected. For advice on site selection based on diversity analysis, see Maxted et al. (2004, 2008b) and Iriondo et al. (2008). For taxa that occur both within and outside PAs, existing PAs should ideally be selected for inclusion in the CWR genetic reserve network. However, a balance will have to be met between ecogeographic suitability of sites and feasibility. In other words, if very little intra taxon genetic diversity is likely to be conserved by enacting active conservation of populations within existing PAs, it may also be necessary to recommend that new genetic reserves are established for other genetically distinct populations.

20 When possible, a range of taxa should be assessed all together to try to ascertain whether it is feasible to establish multi taxa reserves. When focusing on a crop gene pool conservation strategy, this may not always be possible, depending on whether the target taxa are found at the same locations. The identification of multi taxa reserves is most likely to be achieved through the development of national CWR strategies which target a country's entire CWR flora. Therefore, during the development of a crop gene pool in situ conservation strategy, collaboration with organizations responsible for developing national CWR conservation strategies is advised in countries where the target crop gene pool taxa are to be conserved. The potential effects of climate change on populations of the target taxa also need to be taken into account. Considerations include the particular vulnerablity of populations in coastal and high altitude areas, whether there is sufficient intra population genetic diversity and reproductive success in populations to allow adaptation to new conditions, and whether small, fragmented populations with little migration will be able to colonize new sites (Veteläinen et al., 2007). In the absence of detailed studies on individual target taxa, it will not be possible to predict exactly where sites need to be established because a) we will not know whether populations of a taxon will have the ability to adapt to new conditions at current sites, b) whether populations will have the ability to migrate to new sites, and c) if migration occurs, how quickly it will take place and in what direction. However, greater emphasis on habitat protection to prevent and reduce habitat fragmentation and the establishment of corridors between habitat patches to facilitate range shifts of mobile species is likely to be important for many CWR taxa (Jarvis et al., 2008). 3. Prioritize the selected sites. The main criterion for allocating priorities to sites is the conservation of the maximum genetic diversity possible. When assigning priorities for a particular target taxon, the diversity analysis will form the basis of the priority ranking of sites. When the aim is to conserve multiple taxa within the same sites, a balance has to be met between prioritizing those sites that contain the greatest taxonomic diversity and those that contain less taxonomic diversity, but more genetic diversity specific to particular target taxa. Other factors to take into account when assigning priority ranking to selected sites include: land use, potential development pressures (e.g., sites closer to towns and cities may be less secure), presence of invasive species (particularly on islands), level and quality of site management, legal status, potential conflict with existing site management aims and social unrest. A thorough assessment of all factors, both scientific and socio political, must be made and considered when selecting the ideal sites. 16

21 3.0 Application of the methodology to four crop case studies 3.1 Crop case studies: overview and purpose The overarching aim of the AEGRO project is the development and promotion of CWR and landrace conservation strategies as a contribution to an integrated EU agrobiodiversity conservation strategy. To develop and test the CWR in situ conservation strategy methodology and to kick start the integrated EU conservation strategy for CWR, four crop case studies were selected for detailed analysis and subsequent conservation strategy planning Avena sativa (oat), Beta vulgaris subsp. vulgaris (cultivated beets), Brassica spp. (cabbage and other brassicas) and Prunus avium (cherry). The selection of crops was endorsed by experts who are members of the European Cooperative Programme for Plant Genetic Resources (ECPGR) crop networks. Each of these crops is a high priority in economic terms for Europe (Kell et al., 2011), as well as being nutritionally important. Further, in addition to providing a vehicle for illustrating how to achieve in situ CWR conservation, these case studies represent four diverse crops with differing habits, life cycles and reproductive biology a grain, root and leafy vegetable crop and broad leaved tree species; therefore, the lessons learned in conservation planning for these case studies can be applied to other crops sharing similar characteristics within each of these biological and use groups. For each of the crop case studies, an in situ CWR conservation strategy has been developed which has resulted in the identification of CWR genetic reserve sites for high priority taxa within each crop gene pool throughout their European range, with a more detailed strategy developed at national level for Portugal for the Beta case study. Each strategy has been developed in the framework of the standard protocol presented in section 2.0. The following sections are an analysis of the application of the methodology for each individual crop case study. The purpose of the analysis is to provide a brief commentary on the practical application of each of the four steps of the methodology with the aims of: Revealing different perspectives on the application of the protocol by each of the crop case study experts; Investigating ways in which the application of the individual steps may differ between the four crop groups; Scrutinizing the methodology to confirm its applicability to a range of crop groups; Refining the methodology to ensure that it is widely applicable to any crop gene pool and easily understood by all those involved in CWR in situ conservation strategy planning. 17

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