1 Centro de Investigacións Forestais de Lourizán, Pontevedra, Spain
² IRTA Mas Bové, Reus, Spain
³ Departamento CIFOR-INIA, Madrid, Spain

The inclusion of Walnut (Juglans regia L.) in the framework of EUFORGEN Noble Hardwoods Network is based in some of its characteristics. The scattered distribution of the species, in the west European range, the limited extension of the stands, in many occasions as isolated trees, and its precious timber, greatly appreciated for many uses. The main factors affecting the genetic resources of the species derives from the traditional plantation by man resulting in a complex pattern of native (if existing) and man-made stands, and the introgression of these cultivars with native trees. Recently, the use of cultivars of Juglans regia and Juglans nigra are becoming more important. Within the Noble Hardwoods Network, it shares some characteristics factors of the strategies of genetic resource conservation with Chestnut, mainly due the multipurpose objectives of both species. However the differences are quite high, due to the scarce presence and role of Juglans spp. in the forest. Walnut is usually found in stands of limited extension, in pure stands (the presence in Western Europe in mixed broadleaves stands is rare), and the until recently limited importance of cultivars in the silviculture of the species.

Walnut grows as a wild species in mixed broadleaves forest from Southern Europe to India. It is found in the Balkan area (Hungary, former Yugoslavia, Bulgaria, Albany), Caucasus, Asia Minor (Turkey, North Iran and Iraq), central Asia (the North limit in Kyrgyzstan), having its eastern limit in the North of India (Hemery, 1998; Malvolti et al., 1997; Germain, 1999). The natural area of the species is a matter of controversy, and little is known about the native area in Europe, and even in the Balkan area some authors consider it as an introduced species (Malvolti et al 1998). Central Asia and more specifically the mountains placed at West of Himalayan chain in the Cashmere, Tajikistan and Kyrgyzstan are considered to be the centre of origin of J. regia L. Surprisingly in that area there are populations of J. regia with very different characteristics: some of them are formed of high trees with remarkable stem straightness and others include smaller trees that produce big crops of nuts and continuous flower during the growing season. (Hemery, 1998; Germain et al., 1997 and 1999).It has been cultivated since the Greek and Roman times all around the Mediterranean basin, where it is found as scattered individuals or groups of several trees in the borders of agricultural lands, orchards or by the rivers, usually close to human settlements. The species was spread all around south Europe already in 1000 BC (Ducci et al., 1997). As a basis for the genetic resource conservation of the species, we can assume the existence of native trees all around the Western Mediterranean area. This hypothesis has to be deeply studied.

It is a long-lived species, and it is not difficult to find trees of 100-200 years old, and even some 1,000 years old. There are some problems related to natural regeneration due to the fact that the edible nuts are eaten by different animals. Walnut is found in all types of substrates, mainly in fresh, fairly rich, and loose soils, avoiding gypseous, compact, or dry ones. The optimum soil pH ranges from 6.5 to 7.5. It prefers mild to warm-mild climates, as late frost is an important limiting factor that affects both flowering and shoot development. In these climatic conditions, altitude is not an important limiting factor, and it can be found from low elevations up to 2,500 m asl (Himalayas, Morocco) (Leslie and McGranahan, 1988 and 1997).

In Europe, walnut can not be considered as a social species, in the sense that no large monospecific stands are found, as it is usually found as isolated trees with a high spacing and producing a dense cover of the ground, or in small groups of trees.

One of the main characteristics of the species is its multiple use, that has affected the present increase of the distribution area of the species. Walnut is planted for producing both nuts and timber. The ornamental value of the tree is of great importance in all the European range of the species, in parks or in the countryside by the houses. The wood is hard, homogeneous, heavy, with a dark brown heartwood, with a highly appreciated pattern. By steaming, it produces a nice reddish colour or a black and red pattern of great beauty. Due to these characteristics and their easy machining it is very appreciated in carpentry. It is used for veneer, fetching very high prices for furniture. Walnut timber is considered the most valuable timber from the European ones.

The fruit is edible and is highly appreciated for human consumption. As a result, many countries developed selection and breeding programmes for nut production. In Western Europe, new orchards are planted using grafted plants of cultivars issued from French and Californian breeding programmes which have allowed a high improvement of orchard productivity (McGranahan, 1997; Germain, 1999). In Eastern Europe plantations of clonal grafted varieties are not so common.

In general, the European walnut production still largely depends on trees originated from seedlings. During the last 20 years, an important work on seedling selection has been carried out in local populations of J regia throughout Europe. The characteristics of wild walnut trees been described in Bulgaria, Germany, Greece, Hungary, Italy, Poland, Portugal, Romania, Slovenia, Spain, Ukraine have (Germain, 1992, Germain et al., 1983 and 1997; Deaconu and Vasilescu, 1997; Pielko, 1989; Revin, 1989; Solar, 1989).

The trend in plantations and consequently in selection and breeding, is to separate the production of wood from the production of fruit. However, all genetic information issued from walnut breeding programmes is also being used in current programs focused on wood production (FAIR III-CT-96-1887).

The clear separation that exists among breeding programs implies the necessity of including the two objectives in the genetic conservation of the species.

The surface planted with Juglans species has recently increased due to EU directive on afforestation of abandoned marginal lands (EU Directive 2078) and so has the use of interspecific hybrids, that can be found in specialised nurseries (Becquey, 1997).

At present, most of the nuts and the timber in Western Europe come from walnuts growing in the countryside. In France, however, most of them are produced in plantations.

As this species is very sensitive to a number of abiotic and biotic factors, it is possible to suppose that natural selection is a very strong force in walnut populations, particularly in south-western Europe. The most important abiotic factors are autumn frosts, that sometimes lead to tree death and late spring frosts that have an effect on stem form. The main biotic damage factors are fungus attacks. Armillaria mellea, Phytophthora cinamomii and P. cambivora are important diseases affecting the root system and antracnosis, Gnomonia leptostyla causes summer leaf fall. Bacterial diseases are important too. Xanthomonas arboricola pv. juglandis, damages leaves and young shoots in humid and mild climate and after several rainy summers some trees might even die (Becquey, 1997). Erwinia spp should be also considered as it damages bark and wood and although it might not kill the trees produces log value depreciation (Teviotdale et al., 1985; López et al., 1994).

Walnut is an anemophilous species, produces heavy pollen that moves over 100 metres. Although dichogamy is present there is autocompatibility (Germain et al., 1973). These facts and the scattered distribution of the species it is possible to suppose that rate of gene flow is low among stands. The presence of apomixis is reduced to cultivars from Central Europe and it is highly influenced by the weather during pollination period. Some Byelorussian, German and Hungarian selections have shown this phenomenon. In some conditions, up to 70% of apomixis has been described (Loiko, 1989; Germain, 1999).

According with the scheme of Eriksson (1996, 1998), a very strong differentiation of adaptive traits can be assumed among populations located in different ecological conditions. This is due to the action of natural selection, the scant importance of gene flow and the very limited phenotypic plasticity of walnut. However, within a homogeneous ecological area, there are several populations subjected to the same natural selection processes (especially in relation with temperature and diseases) but among which there is no gene flow. Therefore, we can expect an important differentiation among this populations because of genetic drift and inbreeding within each one.

In fact, most authors consider that these SW European populations are adapted to local environments and the first results of provenance tests confirm this hypothesis. For example, in Spain significant differences among populations have been detected in survival, height growth and heat sum to leafing out (Fernández and Pereira, 1997), and in vigour, leaf fall and blight sensitivity (Aleta and Ninot 1997). Some variation in resistance to Armillaria mellea was met in Juglans regia progenies (Desray et al., 1997). The first results of different progeny tests developed in the European project showed a very important heritability for height and phenology (see first report of FAIR-III, CT-96-1887).

Twenty-six naturalised populations from Mediterranean an Asian origins were studied using isozymes (Malvolti and Fineschi, 1987; Malvolti et al. 1997) and it was found that the values of polymorphism and heterozigosity (38.33-46.7; 0.152-0.225 respectively) were lower than those reported for Chestnut.

Malvolti et al.(1997) indicate that wild trees have arisen from genotypes that escaped cultivation but in some areas the contribution of native residual germplasm cannot be excluded. These authors mention that despite the limited level of genetic variability within J. regia, two main factors have to be considered. Firstly, the presence of genotypes adapted to local environmental conditions, as deduced by the variation of F statistics among populations from different areas. Secondly, the occurrence of different alleles in populations from China and Europe suggest the existence of distinguished gene pools.

As a result of exploration and selection in local populations, in many European countries there are several J. regia native germplasm collections. A list of this material and its localisation will be available next year when FAO publishes the information in a specific publication of J. regia genetic resources (Germain, Coord., in preparation).

The main threat to genetic variability, at least in South West Europe, is and was the felling of the best trees for wood production, due to the high price of the timber.

Another threat is posed by the introgression of black walnut (Juglans nigra) or of grafted varieties for nut production. Many crosses are possible within the Juglans genus and interspecific hybridisations are very common. Particularly interesting for wood production are the hybrids J. nigra or J. major x J. regia (Becquey, 1997). Sometimes F₁ hybrids are similar to J. regia specially when trees are still in the nursery but morphological characteristics (such as cataphylar leaf distribution) or isozyme characterisation can be used for identification (Arulsekar et al., 1985; Germain et al., 1993; Hussendörfer, 1999).

Finally, the abandonment of the countryside, a very generalised process, could have a very strong influence in the naturalised populations.

The main genetic resources to be conserved in SW Europe are the various small sized (5 to 12 trees) scattered populations, that appear to be a long standing introduced genetic resource, well adapted to the local environment. These may be considered as the gene resource population for selection of nut or wood varieties. The main South Western European Germplasm collections conserve much more variability regarding nut production than forestry traits and usually the accessions are clonal selections, which means that population variability is not always preserved. However, a question that can be addressed is the genetic basis of these populations. At the moment not much information is available on the amount of genetic diversity or the existence of founder effects in the W European material. In this case, the introduction of new germplasm should be considered. This could be collected from areas where genetic variability is still high, e.g. Central Asia (Hemery, 1998), or from countries where wild populations are widely present as they are the source of walnut timber for European manufacturers, e.g. Greece, Turkey or Iran (Jay-Allemand et al., 1996). This last objective should be clarified by research programs before taking any decisions.

Objectives of Walnut forest Genetic Resources Conservation within the framework of the EUFORGEN Noble Hardwoods Network are based mainly on the multipurpose use of the species, and therefore, could be divided into three main objectives:

1. Conservation of the present genetic resource base, using the different collections of selected trees for wood or nut production with objectives of breeding.

2. Conservation of the present genetic base, taking into account the small size of populations in the European range of the species.

3. Dynamic conservation of the species by creating good conditions for future evolution of populations.

From a European perspective the gene conservation strategy of Walnut should consider the following issues:

1. As in Europe most of the trees are located in the countryside and not in the forest, which definition of population should be applied.

1. How many populations and genetic entries should be included.

2. How should populations be sampled.

3. Which are the priority activities to be developed.

The following methods are suggested:

1. Clonal archives of plus trees and valuable trees for fruit production already selected. As pointed out by Ducci et al. (1997), there are already some collections of great value that could be used for static conservation of genetic resources. Broad collections are currently found in France, Italy, Spain and Hungary.

2. Clonal seed orchards containing grafted plus trees provenance areas with 50 p.t. The promotion of planting with seedlings of such origin is a very effective way to increase genetic variability (Eriksson, 1998) in western Europe at a level that never exist before.

To establish a MPBS, according to the principles developed by Eriksson et al. (1993) and applied by Varela and Eriksson (1995), at least twenty populations of 50 genetic entries each must be chosen. In some situations the population size must be increased.

Progeny tests of 50 trees collected in an ecologically homogeneous area. This can be especially interesting in areas where there is a breeding programme for forestry traits.

It is also necessary to identify methods to transform the progeny test into a conservation plantation, after progeny test evaluation and further stimulation of regeneration are completed.

As was previously mentioned, in this species, the size of populations is quite limited. Therefore, these should be considered in a broad sense, in order to include at least 20 trees. Within these areas, natural propagation has to be promoted in order to increase the size. Due to the economic and social value of walnut management of these populations is the only way to maintain and increase the size of the populations.

Clonal archives of plus trees or clones selected for nut production and seed orchards will be maintained in each country interested in plantations and in genetic improvement.

In order to establish a dynamic conservation programme, populations should be selected throughout the entire European area and distributed among the different climatic zones: eight populations in true Mediterranean (2 in Portugal, 3 in Spain, 2 in Itlaly, 1 in Turkey); two populations in atlantic temperate-humid (1 in Turkey, 1 in Armenia); four populations in atlantic-oceanic (2 in Spain, 1 in France, 1 in UK); and seven in atlantic middle european (1 in France, 1 in Italy, 1 in Germany, 1 in Poland; 1 in Hungary, 1 in Bulgaria and 1 in Romania). Walter climatic classification was used (Allue-Andrade, 1990).

Studies using different genetic markers (maternally and paternally inherited) to asses the migration pathways of the species, the role of native stands in Western Europe, and the mating system in different ecological conditions are needed.

Provenance tests, connected with the previous studies, to asses the adaptive variation of the species, including provenances from the entire distribution area are also required, together with a further evaluation of the interest of infusion of germplasm from SE Europe or Asia in SW Europe.

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