Crop case study Beta L. (including Patellifolia A. J. Scott et al.)

L. Frese, M.A Pinheiro de Carvalho, C. Duarte

Introduction Beta vulgaris L. subsp. maritima Arcang. is considered the ancestor of all cultivated beets. Zossimovich (1939) cited by Boughey (1981) assumed that the cultivated beets were first grown on irrigated fields of ancient Mesopotamia but fossiles supporting this theory were never excavated. There is however historical evidence that the cultivation of beets as green diet was well known to the Greek people in 370 B.C. At that time Theophrastus even described the differences between wild and cultivated forms (Boughey, 1981). Beets cultivated as green salad vegetable are likely the ancestor of garden, fodder and sugar beets, the development of the latter started only 200 years ago. From an evolutionary point of view the sugar beet was developed within a very short time towards a high productive crop. Bosemark (1989) suggested therefore maintaining the adaptability of sugar beet by continuous and systematic incorporation of genetic variability from the closest relative, i.e. Beta vulgaris subsp. maritima, into the elite gene pool. Biancardi (2005) reviewed the objectives of sugar beet breeding and devoted several chapters to resistance breeding. Genetic protection of the sugar beet crop against viruses (Curly Top, Beet Yellows, Beet mosaic, Rhizomania, Beet soil borne virus), bacteria (Bacterial vascular necrosis and rot, Yellow wilt), fungi (Cercospora leaf spot, Powdery mildew, Downey mildew, Fusarium yellows, Root rots such as Rhizoctonia, Phoma, Pythium, Aphanomyces, Sclerotium), Polymyxa, Alternaria, Ramularia, Uromyces), and pests (Cyst and root knot nematode, Aphids, Spinach leaf miner, Sugar beet root maggot, Flea beetle and last not least Spider mites are welcomed by growers and consumer since it helps to keep production costs lower and helps reducing pollution of the environment with fungicides and insecticide residues. To achieve the resistance breeding objectives breeders have used wild beets as a source of novel genes. Steinrücken (2005) anticipates an even growing demand for genetic resources. He argues that the improved breeding techniques based on molecular marker technologies and an improved knowledge of the Beta genome will facilitate the integration of genes from exotic germplasm into the crop and therefore increase the interest in Beta genetic resources and the demand for novel genetic variation.

As a consequence of climate warming growing conditions are changing at accelerated rates and from the evolutionary perspective within an extremely short period. The domesticated species developed from their highly diverse wild ancestors may not always have the genetic variation available needed to cope with these fast changes. The genetic reserve conservation technique (Maxted et al., 1997) recognizes the need for maintenance of the intraspecific diversity which is the natural basis of adaptation of any species to environmental changes and a crucial condition for its survival in rapidly changing environments. A wide genetic basis of wild species related with a crop ensures the evolution of new genes and alleles required for the adaptation of that crop. There are therefore strong arguments to protect crop wild relatives in the natural surrounding where they have developed their adaptive traits as well as traits currently used by plant breeders. The establishment of genetic reserves is thus a service of nature and species conservation to agriculture.

The design, planning and establishment of genetic reserves are technical conditions for the maintenance of intraspecific diversity. A step-wise methodology for the identification of genetic reserve sites for a target crop gene pool was developed by the University of Birmingham within the AEGRO project. The methodology can be accessed online at http://aegro.bafz.de/index.php?id=188. Recommendations for the choice of genetic reserves sites for Beta and Patellifolia (syn. Beta section Procumbentes Ulbrich) within the EU-27 as well as in Portugal and Germany described in the case study are based on this 4-step-methodology.

Application of the four step methodology The case study consists of three parts. As there is no need to repeat step 1 of the methodology for the national level case study, the case study begin with the taxon delineation and continues with EU-27 level case study (Part A) followed by the national level case study (Part B). Step 1: Taxon delineation Information on the distribution of wild species can be derived from various information systems and data sources. Since there is no single globally accepted taxonomic system for Beta three systems were implemented in the CWRIS AEGRO Population Level Information System (PLIS) (CWRIS AEGRO PLIS, 2010). The case study is based on a taxonomic system proposed by Frese. The relation of sections, species and gene pools is presented in Table 1.

The fourth section of the genus (Beta section Procumbentes) has been accepted as the new genus Patellifolia (Scott et al., 1977). Although Patellifolia species are no longer part of Beta they should be treated as wild beets belonging to the gene pool of cultivated beets. This suggestion is well in agreement with the definition of a gene pool published by Harlan and de Wet (1971). The suggestion of Kadereit et al. (2006) to include B. nana Boiss. & Heldr. into Beta section Corollinae Ulbich was also considered by the working taxonomy for Beta shown in Table 1.

Table 1. Beta working taxonomy and assignment of taxa to the primary, secondary and tertiary gene pool of cultivated beets

Gene pool Genus Beta L. Primary Section Beta Transhel

B. vulgaris L. subsp. vulgaris (cultivated beets) Leaf Beet Group Garden Beet Group Fodder Beet Group Sugar Beet Group

subsp. maritima (L.) Arcang. subsp. adanensis (Pamuk.) Ford-Lloyd & Will.

B. patula Ait. B. macrocarpa Guss. Secondary Section Corollinae Ulbrich

Base species B. corolliflora Zosimovich B. macrorhiza Steven B. lomatogona Fisch & Meyer

Hybrid species B. intermedia Bunge B. trigyna Wald. & Kid.

B. nana Boiss. & Heldr. Tertiary Genus Patellifolia A. J. Scott et al. P. procumbens (Smith) A.J.Scott, Ford-Lloyd & J.T.Williams P. webbiana (Moq.) A.J.Scott, Ford-Lloyd & J.T.Williams P. patellaris (Moq.) A.J.Scott, Ford-Lloyd & J.T.Williams

Although the identification of genetic reserve sites for CWR is the main purpose of this case study the taxonomy of cultivated beets is commented here. A future study should consider cultivated forms to facilitate decision to be taken in the field of on farm management. The highly detailed taxonomy of cultivated beets used by many gene banks (“splitter system”) requires intimate knowledge of the meaning of taxonomic names and cannot be used straightforward by sporadic users of information systems such as Global Biodiversity Information Facility (GBIF). In particular the downloaded GBIF file contained a large number and variation of taxonomic names of cultivated forms which have been processed and harmonized along with the wild species taxonomic names.

The taxonomic names used by “splitter systems” were matched to the “lumber system” for cultivated types. The latter was proposed by Lange et al. (1999) who have sorted all cultivated forms into four cultivar groups. This simple and clear classification proposed by Lange et al. (1999) is part of the Frese system used by CWRIS AEGRO PLIS but has a disadvantage: A highly detailed splitter system can be mapped to the lumber system. Mapping in the opposite direction is not possible as long as the accessions classified according to Lange et al. (1999) has not been characterised in detail. An

2 example: Beta vulgaris subsp. vulgaris convar. cicla var. flavescens forma rhodopleura is classified by Lange et al. (1999) as Beta vulgaris subsp. vulgaris culton Leaf Beet group. The “convar. cicla var. flavescens forma rhodopleura” describes a red coloured leaf beet with, while “culton Leaf Beet groups” circumscribes a leaf beet, only.

Not all relationships between taxonomic names used to describe variation in the cultivated species have been elaborated during the preparation of the information basis. In particular the Russian informal taxonomic system has not yet been analysed in detail and mapped to the three systems implemented in CWRIS AEGRO PLIS since work on cultonomy is not part of this case study.

A list of synonyms for taxonomic names contained in information systems and/or described in literature can be found at http://aegro.bafz.de/aegroprod_beta/gm/queryBuilder/findTaxon.seam.

Part A of the case study: EU-27

Step 2: Selection of target taxa According to Ford-Lloyd et al. (2009) genetic reserves should be established if a taxon occurs is less than 10 geographic units. Geographic units are listed in the Crop Wild Relative Information System (CWRIS, 2009). This concept was applied and yielded the following list. Taxa printed in bold letter are naturally distributed within the EU-27 while the other species may be encountered due to sporadic carryover into EU-27 countries.

B. patula B. macrocarpa (4x) B. vulgaris subsp. adanensis B. corolliflora B. macrorhiza B. lomatogona B. nana B. procumbens Smith (=Patellifolia procumbens) B. webbiana Moq. (=Patellifolia webbiana)

B. patula is confined to two islets off the main island Madeira (Pinheiro et al., 2010) which counts for a single geographic unit.

B. macrocarpa occurs in more than 10 geographic units. Until recently it was assumed that the diploid form is mainly distributed in Portugal, Spain, Greece, Israel and Morocco while the distribution of the tetraploid form is restricted to the Canary Islands. This assumption was revised by Villain (2007) who detected diploid B. macrocarpa on the Canary Islands. Tetraploid forms have not been found in the continental part of the distribution area to date as allopolyploid offsprings of B. vulgaris subsp. maritima x B. macrocarpa are extremely rare in the Iberian part of the distribution area. Villain (2007) confirmed Lange and de Bock (1989) who had suggested an allopolyploid origin of the tetraploid type of B. macrocarpa and mentioned the absence of backcrosses between 4x “macrocarpa” with its diploid parental species. She further suggested that the endemism of the tetraploid B. macrocarpa, the morphologically differences between the 2x and 4x type as well as the genetic isolation between allopolyploid type and the ancestral taxa would allow ranking of the 4x type as a separate taxon. The use of different taxonomic names for the 2x and 4x type would also accentuate the fact that B. macrocarpa 4x occurs in five geographic units only (CWRIS, 2009).

The species (2x and 4x type) lives in a very specific niche, i.e. in a dry climate on disturbed often haline soils. In Portugal and Spain large reproducing plant groups occur in sea salt winning areas. Extensively managed salt winning areas where owners tolerate haline vegetation on dams and aside tracks are most suited for the species‟ growth and reproduction. Occurrences with a very limited vegetative growth and a reproduction close to zero have been observed by Frese along tracks in the Cabo de Gata Natura 2000 protected area in 2006. The fact that the diploid B. macrocarpa occurs in more than 10 geographic units (CWRIS, 2009) is thus no evidence for the ability of the species to survive the next 20 to 30 years. A threat assessment study should therefore be performed before B. macrocarpa (2x) is definitely excluded from the priority list.

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B. vulgaris subsp. adanensis belongs to the group of annual wild beets of section Beta distributed in the Mediterranean basin and the Atlantic regions of Spain, Portugal and Morocco. Within the EU-27 the distribution area of this subspecies is confined to Greece (Peloponnesus, and islands Kos, Leros, Samos, Chios, Crete, Rhodos) as well as Cyprus (Letschert, 1993).

P. procumbens The species can be found on Tenerife, Gran Canaria, Hiero, Gomera, Fuerteventura and Lanzarote (Bramwell and Bramwell, 1974).

P. webbiana This species is restricted to Gran Canaria, Tenerife and Fuerteventura (Bramwell and Bramwell, 1974).

P. patellaris occurs in large plant groups on the Canary Islands (personal communication of A. Santos Guerra). Despite this fact, a threat assessment should be performed as the species is perhaps endangered in the continental part of the distribution area. Frese (own observations, spring 2006) reported low number of individuals at sites in Spain (Almeria) as did El Bahloul et al. (2009) for Morocco.

A summary of the target taxa selection process gives Tab. 2.

Table 2. EU-27 priority list for Beta and Patellifolia

Taxon No. of geographic Endemic species Threatened Value for units >10 within within the EU-27? species (red list) breeding 1) EU-27? B. vulgaris subsp. Yes No No Yes maritima B. vulgaris subsp. No No Yes adanensis B. patula No Yes Yes B. macrocarpa 4x No Yes B. macrocarpa 2x Yes Yes B. corolliflora n.a. n.a. n.a. Yes B. macrorhiza n.a. n.a. n.a. Yes B. lomatogona n.a. n.a. n.a. Yes B. trigyna n.a. n.a. n.a. Yes B. nana No Yes Yes Yes P. patellaris Yes No No Yes P. procumbens No Yes Yes P. webbiana No Yes Yes Yes 1) According to Frese et al. (2001) and Schliephake et al. (2010) Step 3: Ecogeographic diversity analysis by species

B. macrocarpa (4x) and B. macrocarpa (2x)

Geographic

Buttler (1977) described the morphology of the annual wild beets and concluded that “… only tepal characters show a geographic pattern and hence are worth taxonomic recognition”. He further divided these wild beets into three types using tepal and ovary lids as distinction criteria. Type 1, named the western group (B. macrocarpa Guss., synonym B. bourgaei Cosson, diploid, connivent thin tepals) is mainly distributed in Sicily, Spain, Portugal, Canary Islands with outposts in the East Aegean region and mainland Greece (Attika), Israel and Jordania; type 2 with connivent spongy tepals in the South Aegean region and type 3, the eastern group with appressed tepals is ranging from Jordan, Israel, Syria, Turkey, the east Aegean Islands to north eastern Greece. He called B. macrocarpa 4x an evolutionary young group, a hypothesis that is now substantiated by Villain (2007). Although also found in “macrocarpa” occurrences in Israel and Jordan type 3 prevails in “adanensis”.

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The 4x type of B. macrocarpa is the only natural polyploid form within Beta section Beta. It occurs on the Canary Islands, only (except for provenances introduced to California by chance). The species was found on Gran Canaria, La Palma, Tenerife, La Gomera (Buttler, 1977) as well as Fuerteventura and Lanzarote (Villain, 2007). Villain described the distribution of the tetraploid and diploid form. The first one can be encountered more frequently in the eastern part of the Archipelago and grows at an altitude lower than 200 m, while the diploid type is more frequent in the western part and grows between 200 m and 400 m, sometimes up to 600 m. Sympatric occurrences of both forms can be found up to 300 m.

The decision of establishing genetic reserves has to be based on the most recent plant survey which is the one of Villain (2007). The distribution of B. macrocarpa (2x and 4x) is shown in Fig. 1 while the range of climatic conditions to which the species is adapted is depicted in Fig. 2. The environment of the Canary Islands is described in more detail in the next paragraph.

Ecological/ environmental data Bramwell and Bramwell (1984) classified the climate and vegetation types of the Canary Islands. B. macrocarpa (4x) is found up to 300 m. It belongs to the semi-desert succulent scrub vegetation zone which has a hot, dry Mediterranean climate. However, locally the environmental conditions can considerable deviate from the regional macroclimate. The north side of all western islands are cooler and more humid. Occurrences described by Villain (2007) were found in areas with an average annual temperature of 16.8 °C and precipitation of 798 mm (Buenavista, Tenerife) as well as an average annual temperature of 19.0 °C and a precipitation of 108 mm (La Olivia, Fuerteventura) (http://www.globalbioclimatics.org/default.htm). The species was found growing on wasteland, on pilings along roadside, and between boulders and gravel along the seaside, in general on red soil, only in a single case on salty soil.

Figure 1. Distribution of B. macrocarpa (2x and 4x). Green symbols indicate occurrences located within protected areas. Due to a wrongly recorded geographic coordinate an occurrence observed at Kairouan (TUN) is mapped in the Gulf of Guinea.

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Figure 2. The major climatic differences within the distribution area of B. macrocarpa. Left diagram: winter rain and high summer temperatures in the eastern part. Central diagram: High summer temperatures, very little winter rain. Right: Warm temperatures, small annual temperature amplitude, winter rain.

Genetic B. macrocarpa is an autogamous annual species (Letschert, 1993). There is growing evidence that the 4x type is allotetraploid and the result of the hybridisation between B. vulgaris subsp. maritima x B. macrocarpa 2x (Lange and de Bock, 1989, Villain, 2007).

Interestingly, the tetraploid type is more frequent on the northern parts of the western islands (La Gomera, Tenerife and Gran Canaria) than on Fuerteventura and Lanzarote. The only pure occurrence of B. macrocarpa 4x seems existing on La Gomera. The ratio of number of 4x plants / total number of plants screened for ploidy level on an island were: La Gomera 9/9, Tenerifa 31/35, Gran Canaria (14/19), Fuerteventura (12/50) and Lanzarote (13/22) (Villain, 2007) which means that the tetraploid type of the species has not only a very narrow distribution area but is also very limited in terms of the total species population size.

Villain (2007) analysed the genetic diversity of the Canarian 2x and 4x types with the help of nine SSR markers. If only loci SB06, SB15 and Bvm3 are considered the tetraploid B. macrocarpa is composed of all alleles of B. macrocarpa 2x plus a single allele that can be found in continental plants of B. vulgaris subsp. maritima.

The polymorphism of the 56 diploid B. macrocarpa plants determined with 9 SSR markers was zero. Only the 77 tetraploid plants showed some diversity with 2 to 6 alleles depending on the locus. The tetraploid plants from Tenerife and La Gomera mostly carried the allele 152 bp of locus SB06 which was found to be absent on Gran Canaria, Fuerteventura and Lanzarote. The allele 165 bp of locus SB06 occurred on the latter three islands, only. The average frequency of both alleles over all islands and 4x plants was 9.87 and 7.24, respectively. Allele bp 150 of SB15 is local to Gran Canaria, Tenerife and La Gomera with an overall frequency of 21.71. Allele 191 of locus 1027 is restricted to Fuerteventura and rare (overall frequency = 2.6), allele 119 bp of Bvm3 is restricted to a single occurrence (Punta del Hildalgo, playa, Tenerife), allele pb 123 of Bvm3 to plants growing at Almaciga, Tenerife and bp 111 of Bvm3 restricted to plants growing at Alojera, La Gomera (Villain, 2007).

B. vulgaris subsp. adanensis

Geographic Letschert (1993) noted that subspecies “maritima” and “adanensis” are morphologically diverging but that the differences were not sufficient to classify “adanensis” as a species as was preferred by Buttler (1977). The operculum shape makes the major taxonomic difference between B. macrocarpa Guss. (depressed) and B. vulgaris subsp. adanensis (Pamukç.) Ford-Lloyd & Williams and B. vulgaris subsp. maritima (L.) Arcang. (convex). In addition, the perianth segment size and the morphology of the maturing fruit separate “adanensis” from annual “maritima”.

B. vulgaris subsp. adanensis forms a distinguishable group of annual wild beet. The distribution of occurrences is shown in Fig. 3 and climatic conditions of the distribution area in Fig. 4.

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Figure 3. Distribution of B. vulgaris subsp. adanensis. Green symbols indicate occurrences located with protected areas.

Ecological/ environmental data Flowering in B. vulgaris subsp. adanensis starts early and is concentrated in the seventh and eighth week after germination. Although known as annual species, a fraction of plants may form fresh branches after seed maturity. Letschert (1993) observed plants on pebbly beaches in Cyprus which seemed to be perennials. The subspecies occurs on beaches but can also be found inland along roadside or as segetal flora in fields. The climatic conditions within the distribution area differ only slightly.

Figure 4. Climatic conditions within the distribution area of B. vulgaris subsp. adanensis

Genetic Letschert (1993) used nine allozyme loci to investigate the genetic variability in taxa of the section Beta. He found higher genetic diversity in B. vulgaris subsp. maritima (He= 0,28) and B. vulgaris subsp. adanensis (He= 0,28) than in B. macrocarpa (He= 0,01). The genetic distance calculated by Nei‟s D coefficient was found to be low between “maritima” and “adanensis” (D=0.11). Occurrences of “adanensis” possessed rare alleles that where not present in any of the analysed “maritima” populations, namely Lap1-3 in IDBBnr. 2199 from Rhodos, Pgi2-3 in IDBBnr. 3105 from Crete. According to the current data set they may fall into the Marshall & Browns‟ category of “rare and local” alleles which Marshall and Brown (1975) felt to be the least important category from the ex situ conservation and germplasm collectors‟ point of view. They argued rightly that it would be extremely

7 difficult to capture these rare alleles during collecting missions. However, if research has already generated information on the existence of rare, locally distributed alleles there is no reason why populations containing these alleles should not be set higher up on the priority list for protection measures. The genetic similarity of provenances within “adanensis” was less (I=0.76) as opposed to “maritima” and “macrocarpa 2x” (both I=0,91). Letschert (1993) concluded from these estimates that allozyme divergence was caused by spatial distance.

Villain (2007) concluded from her analysis of nuclear and chloroplast genetic diversity that B. vulgaris subsp. maritima and B. vulgaris subsp. adanensis are closed related. Almost all analysed occurrences of both subspecies from the same geographic region shared the same chloropastic DNA haplotype. The analysis of the phylogeny using genetic differences with the loci Adh, Cab5 and ITS sustained the hypothesis of a close genetic relationship between the subspecies. Occurrences of “adanensis” and “maritima” always clustered together while B. macrocarpa 2x formed clearly separated clades.

B. nana

Geographic Beta nana Boiss. & Heldr. is endemic to Greece. It has a very limited distribution area (Buttler, 1977, Letschert, 1993). A species survey was conducted by Dale (1980, 1981), and repeated by Frese et al. (2009). Populations of B. nana were found in these mountains: Taygetos, Chelmos, Vardoussia, Giona, Parnassos and Olympos (Fig. 5).

Figure 5. Distribution of B. nana. Green symbols indicate occurrences located with protected areas.

Ecological/ environmental Strid (1995) described the species. It is an inconspicuous, diploid (Franzen and Gustavsson, 1983) plant species, with a small rosette of leaves approximately 10 - 20 cm in diameter, depending on the fertility of the soil. The plant is said to be self-fertile, producing few seed stalks with 10-25 flowers per spike between June and August. The monogerm seedballs dehisce to the ground in the vicinity of the seed plant while still green. Dale (1980) noted that germination of the seed has proved difficult, and assumed that the extremes of temperatures in the natural habitat, leaching of inhibitors, as well as enzymes in the gut of animals may all play a part in successful germination.

The species occurs in the mountains at high altitudes, in limestone substrates and on short open turf at the edge of meadows with late snow. It is the only alpine species of the genus distributed in Greece (Strid, 1995) but not the only alpine Beta species (Buttler, 1977). The general habitat for B. nana is in closed or open depressions with relatively moist soil above 1800 m elevation. The climate at such altitude is cool and moist because clouds often build up around the mountains. The plant populations

8 are mainly found on ranges facing east or northeast, where temperatures are lower during the summer afternoons. Plants also grow in crevices between rocks and in disturbed areas, such as rough tracks or severely grazed open plant communities. Short distance gene flow by seed dispersal (few hundred meters) is facilitated, perhaps, by melt water flows, grazing animals, and birds. Because a few plants were found during the exploration in 2005 growing in cracks of steep cliffs, it is hypothesized that birds may play a role in long-distance dispersal by depositing undigested seeds (Frese et al., 2009).

The prostrate growth habit protects the head of the storage root from being damaged by grazing animals (Dale, 1980, 1981). It is possible that a certain degree of grazing may keep the associated flora short, thereby promoting the survival of the species. Except for a very sheltered site at Mount Parnassos, all sites proved to be grazed to various degrees, primarily by goats. Estimates for the risk of genetic erosion within an occurrence were fluctuating around 100 on a scale of 0 (=no risk) to 200 (=very high risk). Especially on Mount Olympos, the species had reproduced well at several sites within the surveyed area. The overall occurrence sizes ranged from more than 1000 individuals on Mount Olympos to a few individuals on Mount Taygetos (Frese et al., 2009).

Genetic Little is known about the patterns of genetic variation in B. nana. Phenotypic variation observed by Dale (1980) in the natural habitat was low within and between populations. Allozyme patterns of B. nana have been analysed by Nagamine and Ford-Lloyd (1989) who found five unique and invariant alleles compared to a range of other species investigated in the same study indicating a unique phylogenetic position of B. nana within the genus. Shen and co-workers (1998a, 1998b) and earlier studies (Jung et al., 1993) showed that B. nana is more closely related to the section Corollinae than to any other section, although a clearly separated group when using RAPD banding patterns. Like B. nana, the other Corollinae species occur at high altitudes and are part of the secondary genepool in relation to cultivated beet (Buttler, 1977).

Patellifolia procumbens, P. webbiana and P. patellaris

Geographic The genus Patellifolia (syn. Beta sectio Procumbentes) consists of three species, namely the tetraploid P. patellaris and diploid species P. procumbens and P. webbiana. Bramwell and Bramwell (1984) published a determination key and a description of the three species. Among other morphological traits the authors used leave characters to distinguish the species. The leaf shape and size can vary depending on the environmental conditions and age of the plants impeding the species determination. The distribution of P. procumbens and P. patellaris is shown in Fig. 6 and 8.

Figure 6. Distribution of P. procumbens. Green symbols indicate occurrences located with protected areas. Two locations are errors. The geographic coordinates of the genebank was assigned to the Bulgarian accession and the dot in Lybia was produced by wrongly documented geographic coordinates. P. patellaris is not a priority species.

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Figure 7. P. patellaris (syn. B. patellaris) and P. procumbens (syn. B. procumbens)

P. patellaris (Fig. 7, left) is said to be an annual procumbent plant forming a canopy of up to 60 cm height. The leaves are triangular-ovate and usually cordate. The inflorescence is lax with leafy bracts. Cymes are one to three flowered and the fruits are usually single and globular. It is easy to distinguish P. patellaris from P. procumbens if the plants agree with the excellent drawings published by Vilmorin (1923).

Since P. patellaris is tetraploid and 4x plants are generally classified as P. patellaris. There is an interesting “variant” of P. patellaris called Beta campanulata Coss. (synonym) which was found by H. Knoche on Fuerteventura, La Matilla in 1915 and Agadir (Algeria) by M. Maire (both cited by Vilmorin, 1923). The mature fruit has a cup-like shape.

P. procumbens (Fig. 7, right) is a very variable, procumbent and perennial plant. Stems are herbaceous. Leaves are long-petiolate, hastate or sagitate, ovate to deltoid, the margins remotely sinuate-lobed. Flowers are solitary or in groups of 2-3.

The species is distributed in the northern coastal region of Tenerife (Taganana to Teno) and locally very common on the south coast at El Medano and Los Cristianos. It can be found on the north coast of Gran Canaria from Las Palmas to Galdar, at Playa de Jinamar and Telde as well as on Hiero at La Palma and on La Gomera locally in coastal areas. On Fuerteventura it occurs in the southern region from Gran Tarajal to Jandia and on Lanzarote on the northern coast at Playa de Famara and Arrecife.

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Figure 8. Distribution of P. procumbens. Green symbols indicate occurrences located with protected areas.

P. webbiana is very similar to P. procumbens. The more pronounced linear, lanceolate shape of the leave is the major morphological difference. Curtis (1968) was the first raising doubts on the correct ranking of “webbiana”. Wagner et al. (1989) using isozymes assessed the genetic relationship between both species and concluded that P. webbiana may be a variant of P. procumbens, only.

The species was found on Gran Canaria on the northern coast at Cueste de Silva, Guia, and from Agaete to San Nicolas. The existence of P. webbiana on Gran Canaria has been verified by A. Santos Guerra in 2008 (personal communication). Findings were reported for Tenerife at the Puerto des Cruz, on Fuerteventura in the central region at Betacuria, Pajara and Puerto Rosario.

Figure 9. Distribution of P. webbiana. Green symbols indicate occurrences located with protected areas.

Ecological/ environmental data P. procumbens can be found up to 300 m altitude. The species can be encountered in different habitats such as coastal areas, ruderal, disturbed inland sites such as roundabouts, road margins, garbage place or abandoned gardens.

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Only P. patellaris occurs in two different climates (compare Fig. 2, diagram for Tenerife and Almeria).

Genetic P. webbiana and P. procumbens are compatible species forming hybrids easily. There is no further information on the genetic distance between the three Patellifolia taxa or the within species genetic diversity.

B. vulgaris subsp. maritima (no priority)

Letschert (1993) and Villain (2007) have shown that the self-incompatible sea beet is highly diverse as compared to the prioritized self-compatible species B. patula, B. macrocarpa (2x and 4x) and B. vulgaris subsp. adanensis. These species are potential donors of novel genes for beet breeding. However, in practice breeders use B. vulgaris subsp. maritima more intensively than B. patula, B. macrocarpa and B. vulgaris subsp. adanensis. One could argue that genetic reserve for the priority species should be established to protect the species as such. If solely priority species are protected according to the genetic reserve concept then only a fraction of the genetic diversity of section Beta would be actively managed.

In forestry, sites for the establishment of gene conservation units are not primarily selected on the basis of criteria such as endemism, rareness or threat of extinction. It is rather the genetic diversity as such which forest genetic resources experts are going to manage within a European network of dynamic gene conservation units (EUFORGIS 2010). There are reasons why this concept should also be taken into consideration for conservation of selected occurrences of wide-spread, non-threatened species such as the sea beet, B. vulgaris subsp. maritima or P. patellaris.

The distribution area of the sea beet is illustrated in Fig. 10 and shows that the wild species is adapted to many different habitats. A comparison between collecting sites described in recent publications (Fievet et al., 2007, Andersen et al., 2005) with those registered in the International Database for Beta (IDBB, 2010) proof that the species survives well in its natural habitat. Visits of known growing sites in France, Germany and Denmark by Frese and Poulsen in 2008 have shown that the species forms large, old and over a period of 30-40 years stable occurrences. There are therefore good reasons to assume that the sea beet is not threatened. Nevertheless, from the genetic reserves users‟ point of view the active management of selected occurrences is of interest, in particular if these occurrences are known donors of traits used in crop enhancement programs. The following chapter provides information on occurrences either of interest to breeding or known for their high genetic diversity.

12 Geographic

The distribution of the species is shown in Fig. 10.

Figure 10. Distribution of B. vulgaris subsp. maritima. Green symbols indicate occurrences located with protected areas. This is an overview map. Maps can be produced for individual countries by CWRIS AEGRO PLIS or particular regions within countries.

Ecological/ environmental data The subspecies is widely distributed and accordingly adapted to a wide range of environments. A detailed description of the environmental conditions is beyond the scope of this document. The methodology required for the identification of occurrences representing the full range of ecogeographic diversity of the distribution area with the objective to capture the full range of adaptive variation within the sea beet is currently developed and tested by the AEGRO project partner 9, the Juan Rey Carlos University at Madrid.

Although “maritima” grows in general in coastal areas there are an increasing number of reports on inland occurrences of the sea beet. The typical and natural habitat is coast line, in particular the small belt between dunes and high tide. This linear habitat allowed the sea beet to fan out in a seemingly enormous area. In fact, the number of countries located in the distribution area is high but the area actually colonized by the sea beet is just a narrow band along the coasts of these countries. The sea beet is a colonizer. Plant groups are established in one year may go extinct during the winter storms next year. Along the coastline seed and pollen are exchanged between adjacent populations (see Fievet et al. (2007) for a detailed gene flow analysis). The dynamics of foundation, extinction, and geneflow result in differences between occurrences but there is no clear geographic pattern at the regional scale as was shown by Letschert and Frese (1993) using Sicily as a model region.

13

Figure 11. B. vulgaris subsp. maritima is adapted to a very wide range of climatic conditions.

There is however clinal variation over long distances such as vernalisation requirement and day length reaction which determine the flowering time (short in the east Mediterranean area, very long in Ireland) and the reproductive success (Letschert, 1993, van Dijk and Boudry, 1992). It occurs in areas characterised by winter rain and hot and dry summers. The climate diagram from Waterford, Ireland, a site with lower temperatures and high amount of rainfall is depicted on the left side of Fig. 11 to illustrate the northwest European growing conditions of the subspecies while a climate diagram from Greece is shown on the right side.

Genetic Letschert (1993) used 11 allozymes to describe the genetic differentiation between occurrences within the Mediterranean and Atlantic part of the distribution area. He concluded that most of genetic variation detected with these allozymes is more or less evenly distributed. The occurrence of the allozym Acp1-2 in most of the Atlantic occurrences and the absence of Acp1-2 in all samples from the Mediterranean area indicates the existence of Atlantic and Mediterranean genepool. A higher genetic diversity was detected in southeastern and central Mediterranean area where also rare alleles were found (Lap1-5, Mdh1-1, Acp1-7).

The set of germplasm analysed by Villain (2007) covered a similar area. Almost none of her Beta accessions are identical with the material described by Letschert. Villain (2007) used chloroplast DNA and the genetic diversity of four introns to describe haplotypes in the set of subsp. maritima accessions as well as 9 SSR markers to describe nuclear genetic diversity. The results are similar to those of Letschert. B. vulgaris subsp. maritima was divided into an Atlantic group including accessions from the coast of Morocco and a Mediterranean group including all accessions from the eastern coast of Spain. The Strait of Gibraltar likely separates the Atlantic from the Mediterranean genepool. The SSR marker analysis showed a slightly lower genetic diversity in the Mediterranean genepool (He= 0,722 versus He= 0,734) however without being statistically significant. She grouped accessions into three hypothetical set (Atlantic region, western Mediterranean, and oriental region) using the data of a phylogenetic study. The AMOVA of molecular variance of chloroplastic DNA showed that 82% could be explained by variation within the groups and 18% between groups. As the variance between groups is statistically significant, the existence of three large geographic groups between which geneflow is or was reduced in the past is very likely.

It can be concluded that three large, stable and genetically variable provenances would be sufficient to maintain the chloroplastic and nuclear genetic diversity of B. vulgaris subsp. maritima: one on the Atlantic coast, one in the west and one in the east Mediterranean region. But which of the many occurrences is to be selected?

The main objective of genetic diversity conservation is the maintenance of diversity as such. DNA- marker diversity can be applied to describe intraspecific diversity. The choice of sites and the occurrences living in these sites can be based on genetic distance measures, knowledge of genetic boarders, and genetic peculiarities such as rare and local DNA-marker alleles. The breeder, as one of the potential and most important users of genetic reserves, is interested in the maintenance of useful 14 traits and in the evolution of new genetic variants of these traits. The emergence of new variants may be promoted by the maintenance of occurrences in the natural environment where the trait was detected.

Occurrences of B. vulgaris subsp. maritima with traits already used in plant breeding or of potential interest to plant breeders are listed in Tab. 3. It seems that more than a single trait of interest can occur within a sector of the distribution area. In the coastal zone of the Somme estuary and the coastal region northeast of St. Valery-sur-Somme plants with BNYVV and Pythium ultimum resistance as well as cytoplasmic genetic variants can be found. In Italy, the Po estuary is known as the origin of Cercospora beticola resistance, the Holly source of BNYVV resistance and homes P. ultimum resistant plants as well.

Letschert (1993) and Villain (2007) have shown that the genetic diversity as described with allozyme and DNA-markers is rather evenly distributed within B. vulgaris subsp. maritima. Due to a lack of comprehensive evaluation data it is not known whether useful traits show a more structured distribution pattern with useful traits are confined to a particular area.

Table 3. Occurrences of B. vulgaris subsp. maritima containing disease resistance.

Country District Site Latitude Longitude Trait DNK Kalundborg Kalundborg 55°40‟48,96 „‟ N 11°03‟54,15‟‟ E Rizomania Fjord resistance (GRIN query) FRA Amiens Baie de Somme 50°11‟54,25‟‟ N 1°30‟38,91‟‟ E Rizomania resistance Büttner et al. 1997, Frese (documents available at the JKI, 2009) FRA Montreuil Étaples-sur-Mer 50°08‟17,31‟‟ N 1°38‟16,25‟‟ E Cytoplasmic male sterility, type E2, G (Fénard et a al., 2006, Ducos et al., 2001) FRA Amiens Baie de Somme 50°12‟03,01‟‟ N 1°30‟50,22‟‟ E Phythium ultimum, Luterbacher et al. (2000) and IDBB query. ITA Rovigo Porto Levante 45°02‟56,76‟‟ N 12°21‟53,86‟‟ E Cercospora beticola and Rizomania resistance (Stevanato et al., 2001) ITA Veneto Chioggia to 45°12‟24‟‟ N 12°15‟16‟‟ E Phythim Conche ultimum resistance, Luterbacher et al. (2000) and IDBB query. GRC Crete Tsoutsouros 34°59‟10,82‟‟ N 25°17‟21,42‟‟ E Cytoplasmic male sterility, type S4 (Lind- Halldén & Halldén,

15 1995) ESP Almeria Albox 37°23‟23,47‟‟ N 2°08‟53,63‟‟ W mtDNA, type T (Fénard et al., 2006) FRA Corsica Abbartello 41°41‟53,34‟‟ N 8°50‟30,14‟‟ E Aphanomyces cochlioides resistance, Luterbacher et al. (2000) and IDBB query PRT Mafra Ericeira bay 38°57‟30,08‟‟ N 9°24‟55,17‟‟ E Phythium ultimum, Luterbacher et al. (2000) and IDBB query

In outbreeding species the fraction within occurrence genetic variation is generally larger than the between occurrences genetic variation. If this is the case for the sea beet all useful traits can be detected through evaluation of many plants from a single region. Fig. 12 demonstrates an intensively evaluated occurrence. The evaluation data were extracted from the IDBB (http://idbb.bafz.de/- CCDB_PHP/idbb/, searched on 01 September 2009). Only the conservative scores, i.e. the higher scores for disease resistance, were chosen, if the germplasm had been evaluated twice or more for the same trait.

AC 10 BNYVV 8 BMYV 6 4 RS 2 BYV 0 UNIVERSALSCORE

PU CB

EB DSS

Figure 12. Hallands Väderö is an island located close to the Swedish coast and north of the Danish distribution area of B. vulgaris subsp. maritima. The sea beet occurrence was sampled in 1956. Since then the germplasm is maintained ex situ and survived well at the natural site which is part of the Natura 2000 network (Site code SE0420002). Abbreviations: AC = Aphanomyces cochlioides, BMYV= Beet Mild Yellowing Virus, BYV = Beet Yellowing Virus, CB = Cercospora beticola, DSS = Drought stress tolerance, EB = Erysiphe betae, PU = Pythium ultimum, RS = Rhizoctonia solani, BNYVV = Beet Necrotic Yellow Vein Virus. The term “universal score” is described at http://idbb.bafz.de/CCDB_PHP/idbb/Help.html.

16 Step 4: Selection of target sites by species

B. macrocarpa (4x) and B. macrocarpa (2x)

The detailed genetic study of Villain (2007) allowed the determination of occurrences containing together all alleles of 9 SSR markers (Tab. 4). Seven locations are required to achieve the complete protection of these alleles. The respective sites are recommended as genetic reserve candidates. In the light of the new information on the structure of genetic diversity of B. macrocarpa the establishment of additional genetic reserves for B. macrocarpa (2x) on the Iberian Peninsula is recommended although B. macrocarpa 2x is not part of the European priority list.

Within the set of occurrences listed in Tab. 4 GCFEL plays a prominent role as it combines alleles either only occurring in plants from the western or eastern islands. The other six populations complete the allelic set.

As the genetic diversity within B. macrocarpa is very limited and distributed between occurrences rather than within occurrences, additional sites on the continental part of Spain are recommended as genetic reserve candidates. Villain (2007) again provides data and analysis that can be used derive recommendations. She investigated continental (PRT and ESP) occurrences of the species with 11 SSR markers and found three distinct groups. The first group occurs more frequently on inland sites than the second group while the third group grows on highly saline sites.

Table 4. Allelic diversity observed at nine SSR marker loci. Shown are seven polymorphic loci of B. macrocarpa 4x plants. All B. macrocarpa (2x, 4x) distributed in the region of the Autonomous Community of the Canary Islands share all alleles of loci Gaa1 and SB04 in common.

Locus Allele Contained in all 2x plants

LO

FCOT LRIO GCFEL TALM THID TLOS GOA SB06 152 + + 158 + + + + + + + 162 + + + + + + 165 + SB07 263 + + + + + + 264 + + + + + + 267 + + + + + + SB15 142 + + + + + + + 144 + + + + + 150 + + + + 1027 175 + + + + + + + 191 + 201 + + + + + + 202 + Bvm3 100 + + + + + + + 111 + 117 + + + + 119 + 121 + + + 123 + Caa1 128 + + + 152 + + + + + 156 + + + + + + + 159 + Gtt1 115 + + + + + + + 121 + + + + + Cummu 1 17 19 21 22 24 25 26 lative 5

17

Three sites are recommended as genetic reserve candidate with the following arguments. The occurrence at Santa Pola (Alicante, Spain) showed an average number of alleles of 1.09, only, but contains a rare haplotype. Villain detected this haplotype when analysing the mitochondrial diversity of B. macrocarpa 2x with four minisatellite markers. The site San José, Almeria, Spain represents the third plant group distinguished by the SSR marker analysis and is geographically separated from the Fuseta plant group. The occurrence at Fuseta (Olhao, Portugal) shows the highest number of alleles of all continental B. macrocarpa 2x occurrences. A summary of the recommendation gives Tab. 5.

Table 5. Location of the seven occurrences where a genetic reserve for B. macrocarpa (4x) and B. macrocarpa (2x) should be established.

Occurrence code Location Protection status of the site (see Villain, 2007) B. macrocarpa (4x) FCOT Spain, Fuerteventura, El Cotillo Probably outside Natura 2000 LRIO Spain, Lanzarote, Mirador del Rio Natura 2000, to be checked GCFEL Spain, Gran Canaria, San Felipe Probably outside Natura 2000 TALM Spain, Tenerife, Almaciga Natura 2000, to be checked THID Spain, Tenerife, Punta del Hildalgo, Natura 2000, to be checked playa TLOS Spain, Tenerife, Playa Los Silos Probably outside Natura 2000 GOALO Spain, La Gomera, Alojera Probably outside Natura 2000 B. macrocarpa (2x) SAN Spain, Alicante, Santa Pola Natura 2000, to be checked JOS Spain, Almeria, San José Natura 2000, to be checked FUS Portugal, Olhao, Fuseta Corinne Code C23000010

B. vulgaris subsp. adanensis The species has a limited distribution area and is closely related to B. vulgaris subsp. maritima. Hence any recommendation for the establishment of a genetic reserve should take in account that most of genetic diversity of “adanensis” is also present in “maritima”.

The currently available information on rare and local alleles in “adanensis” would justify the establishment of two genetic reserves (Tab. 6).

Table 6. Candidate sites for genetic reserves of B. vulgaris subsp. adanensis.

Country Province, location Protected status of the site Greece Rhodos, Kalavarda ? Greece Dodekanisos, Xerokampos GR4210010

B. nana B. nana is considered a rare but not threatened species requiring no specific conservation measures (Strid, 1995). However, species living in alpine regions are particularly prone to extinction risk from climate change (Parmesan and Yohe, 2003, Grabherr et al., 1994). B. nana is a highly specialised species of the alpine regions of the Greek mountains. Reproduction and migration mechanisms of the species have not been investigated, nor are demographic processes understood; thus the impact of climate change on the ecological niche of the species and its long-term population viability cannot be predicted. Moreover, it is not fully understood how the genetic variation is distributed within a population, among populations within a region, or among regions; and to what extend gene flow between populations within a region and among regions occurs (Panella et al., 2010). Hence, there are good reasons to establish at least three genetic reserves that is actively managed and monitored. It would ensure that genetic erosion with the species is detected early enough for the initiation of species rescue plans.

The distribution area on Mount Olympos would be most suited for the establishment of a genetic reserve because the sites are already located within or close to the Nature Park. Two further sites, one on Mount Parnassos and a second on Mount Giona, would also be a possible choice. The Vathia Lakka site located on Mount Giona could easily be fenced by a shepherd and grazed by sheep or

18 goats in a controlled manner favouring reproduction of the species (Frese et al., 2009, Frese et al., 2010).

P. procumbens and P. webbiana As long as the information on the distribution pattern of genetic diversity within the Patellifolia species is very scarce. The following sites (Table 7) are geographically distant or isolated a may harbour a unique fraction of genetic diversity of the species.

Table 7. Candidate sites for genetic reserves of P. procumbens

Country Province, location Protected status of the site Spain La Gomera, Hermigua ? Spain La Gomera, Playa do Santiago Protected site Spain El Hierro, Pozo de Sabinosa Protected site Spain Santa Cruz de Tenerife, San Cristóbal Protected site des La Laguna

P. webbiana is very rare. A. Santos Guerra (personnel communication) confirmed the existence of the species at La Isleta, Gran Canaria, a protected site. The location is therefore recommended for the establishment of a genetic reserve.

B. vulgaris subsp. maritima The North Atlantic Beta occurrences have been characterized a vast storehouse of germplasm that can be incorporated easily into the crop. Doney (1992) further noted that the greatest genetic variation was found in large, old, undisturbed populations which may have accumulated many stress resistance and growth genes that may be useful and necessary for the future of beet breeding and crop production. Based on the analysis of morphological traits he concluded that a spatial distance of 25-50 km provides sufficient isolation to induce a significant shift in gene frequency. This conclusion agrees with the finding of Fievet et al. (2007) who investigated the genetic diversity of the subspecies with SSR marker. Letschert and Frese (1993) using morphological traits have shown that occurrences distributed along the coast line of Sicily differ, however without a clear geographic pattern. As long as it is not known how the genetic diversity of the sea beet is spatially distributed a selection of growing sites representing this diversity is difficult.

It is however possible to establish gene conservation units in areas such as the Somme estuary, at Kalundborg Fjord and in the Po estuary to specifically maintain genetic variation for resistance traits (see Tab. 8). The occurrence in the Po estuary is known since the early 1910ies, the one at Kalundborg Fjord since 1950ies and in the Somme estuary since the 1970ies. The occurrences at all three sites are old, large and stable and recommended as candidate sites.

Table 8. Recommended sites for genetic reserves for B. vulgaris subsp. maritima

Country Province Site designation Protection status of the site DNK Sjælland Kalundborg Fjord CDDA National code 5653, wildlife reserve. Probably not congruent with the BVM provenance FRA Picardie Somme estuary Probably not protected at the sites where BVM occurs ITA Rovigo, Ferrara Po estuary GIS analysis required to locate BVM within protected areas

19 Part B of the case study: national level (Portugal and Germany)

Steps 2, 3 and 4 are described in the following paragraphs for Portugal and Germany, separately. Each national-level study starts with a description of the species occurring in the country. Then taxa are prioritized, the geographic distribution of each species and the diversity of ecological and environmental conditions of their distribution areas are described, information from genetic analysis reviewed and discussed and finally several sites qualifying for the establishment of a genetic reserve are recommended.

Portugal

Step 2: Selection of target taxa for the detailed national-level case study

Portugal, mainland and Archipelago of Madeira Up-to-date information was provided by a national Portuguese research project performed by the Instituto Nacional de Recursos Biológicos (INRB) (see C. Duarte, 2nd AEGRO project meeting report and Table 9). A recent inventory of the species distributed on the Archipelago of Madeira was produced by Pinheiro de Carvalho et al. (2010) within the framework of the AEGRO project for Madeira in the year 2008.

Within Portugal the following species can be found: B. vulgaris subsp. maritime (BVM), B. macrocarpa Guss. (2x type) (BM), B. patula (BP) as well as P. procumbens (PPR) and P. patellaris (PPA). B. vulgaris subsp. maritima, B. macrocarpa occur on the Azores, the Madeira Islands, and Portugal mainland, B. patula is an endemic species of the Archipelago of Madeira. Beta vulgaris subsp. vulgaris is cultivated Portugal. Patellifolia procumbens: is endemic to the Macaronesian archipelagos of Madeira, Canaries, and Cape Verde while Patellifolia patellaris can also be found in the mainland of Portugal. A priority list for the species is given in Table 9.

Table 9. Results of recent field visits and herbarium search for Beta and Patellifolia occurrences

Species Collected specimens Herbarium Subtotal (2007-2008) specimens B. vulgaris subsp. maritima 55 110 165 B. vulgaris subsp. vulgaris 8 8 B. macrocarpa 9 6 15 P. patellaris 3 3 Total 64 127 191

Although a rich genetic heritage of wild B. vulgaris and related species still exists in Europe, many of the natural habitats where these plants occur are under threat. A first assessment of the conservation status of species implemented by the Instituto Nacional de Recursos Biológicos (INRB) has shown that the survival of B. vulgaris subsp. maritima species seems not to be endangered in the mainland part of Portugal. It is relatively common and widespread occurring in natural habitats like cliff coasts, sand beaches and salt marshes, and often also in ruderal sites, and along roadsides. B. macrocarpa mainly occurs in salt marshes and saline soils. The risk of genetic erosion within the latter species is enormous.

20 Table 10: Prioritized taxon list

Country Taxon Value as gene Threat according to national list National priority donor Portugal BMV Yes No Low BM Yes No, but only few populations occur High BP Yes No, although endemic to Madeira High with a very limited distribution area PPR Yes No, but only few populations occur High PPA Yes No, but only few populations occur High

Step 3: Ecogeographic diversity analysis by species

The results of a recent inventory of Beta and Patellifolia species in mainland Portugal is summarized in Table 9. In total 191 occurrences were found and georeferenced occurrences plotted on topographic (Fig. 13) maps and thematic maps (Fig. 14) (Duarte, 2008). The distribution of the taxa was also described by Frese et al. (1990).

Portugal, mainland

B. vulgaris subsp. maritima

B. vulgaris subsp. maritima is mainly distributed along the coast. As noted by Frese et al. (1990) the species does not occur along sandy beaches such as those existing south and north of Lisboa. However, where the long sandy beaches are interrupted by river deltas depositing clay or loamy soils B. vulgaris subsp. maritima plants can occur in large numbers. All sites were located in the coastal area at low altitudes (0 to 110 m) while Duarte (2008) also detected occurrences in inland areas at higher altitudes. Sites with 500 and more flowering plants were observed by Frese et al. (1990) at: Ericeira Bay, Figueira da Foz, and Alcochete.

Ecological/ environmental data

Duarte (2008) plotted occurrences on maps showing the mean annual temperature (Fig. 14) and mean annual precipitation. The species can be encountered in extremely dry and warm areas in the south of mainland Portugal and cool and wet areas in the north. Mainland Portugal was divided by Thran and Broekhuizen (1965) into to two agroecological subclimatic types, namely 50a / 50 and 51. Subclimate 50a has its northern limit at the town of Areosa and the southern limit at Figueira da Foz. Subclimate 50 starts at this town and extends south to the town of Sines from where subclimate 51 extends to the whole south western and southern area of Portugal. Subclimate 51 is described as warm in summer and warm in winter. The mean annual temperature is 17 °C. The mean annual precipitation amounts to 650 mm. The rainfall between November and January totals to about 300 mm. Subclimate 50/50a can be divided into a warmer (annual temperature 15-17 °C), drier region south of the 40s latitude and a cooler region (50a, annual temperature 14.5-15.5 °C) north of it.

Figure 13. Distribution of B. vulgaris subsp. maritima

The southern part (50) is moderately wet, fairly mild in summer and warm in winter. The northern part (50a) is wet, mild in summer and warm in winter. Depending on the subclimate the mean annual 21 precipitation is 700-900 mm (subclimate 50) and 1000 mm (50a), respectively. The major part of the rain falls in autumn and spring (November to January 300 mm). At the local level Frese et al. (1990) observed during their germplasm collecting mission considerable deviations from the basic subclimate. A very humide microclimate was encountered at Ericeira Bay, Vila Nova de Milfontes, and Praja de Zambujeira.

Figure 14. Occurrences of B. vulgaris subsp. maritima compiled by Duarte (2008) and plotted on thematic maps. Left: annual mean temperature. Right: annual mean precipitation.

B. macrocarpa

On the west coast of Portugal cool and moist Atlantic winds considerably decrease the temperature at the seaside while the temperature remains high several kilometres inland. Decreasing temperatures in the late afternoon along with moisture brought in by the Atlantic wind frequently leads to the formation of fog which was particularly noticeable in the early evening in estuaries and bays sheltered by steep cliffs. The fog remained over night and kept the canopy wet until the late morning. Wild beets heavily infested by mildew were observed in these sites.

B. macrocarpa occurs at low frequency in the area between Lisboa and Setubal and at higher frequency in the coastal region of southeast Portugal (Fig. 15). The largest group of flowering plants with approximately 1000 individuals was found at Tavira on the area of a saltern (Frese et al., 1990).

Figure 15. Distribution of B. macrocarpa

22 Ecological/ environmental data

The species is confined to dry and warm areas as can be seen on Fig. 16. Frese et al. (1990) noted that except for a single case B. macrocarpa occurred always in the area of salterns, i.e. on haline soils. In salt marsh areas and barrier islands the soil type “Solonchaks” can be encountered (PH&P, 2000b).

Figure 16. Occurrences of B. macrocarpa compiled by Duarte (2008) and plotted on thematic maps. Left: annual mean temperature. Right: annual mean precipitation.

P. patellaris

Patellifolia patellaris is a very rare species in mainland Portugal. Occurrences have been reported for a coastal area west of Sesimbra (Fig. 17). As explained above the environmental condition of these few sites may differ considerably from subclimate 50 to which the site belongs to. A description of the ecological conditions of the area would require a survey mission.

Figure 17. P. patellaris occurrences

23 Archipelago of Madeira

The cultivated B. vulgaris L. subsp. vulgaris Leaf Beet Group, the closely related wild beets B. vulgaris L. subsp. maritima (L.) Arcang. and B. patula Aiton and the very distantly related Patellifolia procumbens (C.Sm. ex Hornem) A.J. Scott, B.V. Ford-Lloyd & J.T. Williams and P. patellaris (Moq.) A.J. Scott, B.V. Ford-Lloyd & J.T. Williams (Press, 1994; Jardim and Sequeira, 2008) occur on the archipelago of Madeira. The distribution of these species on the main island, Madeira, was surveyed in the year 2008 and the result is shown in Fig. 18.

Figure 18. Relative abundance of B. patula and associated Beta/Patellifolia species on the islet Ilhéu do Desembarcadouro

B. patula and associated Beta/Patellifolia species

Geographic data

The archipelago of Madeira is located in the Atlantic Ocean 630 km west of North Africa and 900 km southwest of the Iberian Peninsula. The region of Ponta de São Lourenço with its islet Desembarcadouro forms the eastern part of the main island. Southwest of the island Madeira the Desertas Islands consisting of Ilhéu Chão, Deserta Grande und Bugio are located at visibility range. On Madeira B. patula is confined to the islet of Desembarcadouro at the Ponta de São Lourenço. Desembarcadouro is an uninhabited islet with a length of 1.95 km and a width of 0.43 km in its largest and 0.06 km in narrowest part. In the Desertas Islands, the species is confined to the islet Ilhéu Chão, also an uninhabited area of approximately 0.5 km2. The access to this table-like shaped island raising about 80 m above sea-level is difficult. It is the most northern and the smallest of the Desertas Islands, located about 11 nautical miles southwest from Ponta de São Lourenço with its islet Desembarcadouro. The species B. patula, B. vulgaris subsp. maritima and P. procumbens occur on the Desembarcadouro islet and on Ilhéu Chão (Tab. 11 and 12).

The Ilhéu Chão has a unique landscape compared to the other Desertas islands. It is completely flat without relief barriers which could shape the spatial distribution pattern of B. patula. Nevertheless, wild beets (Beta / Patellifolia) have been found on a single site, only, where all three species, i.e. B. patula, B. vulgaris subsp. maritima and P. procumbens were encountered.

24

Table 11. This table presents the preliminary results of fieldwork in Ilhéu Chão, with the number of specimens of wild beet species counted and sampled.

Species Location Site Individuals Samples taken counted B. patula Ilhéu Chão Site 1 134 134 B. vulgaris Ilhéu Chão Site 1 42 42 subsp. maritima P. procumbens Ilhéu Chão Site 1 22 22

The highest number of wild beet plants was detected in the Desembarcadouro islet (Tab. 11). In these areas the wild beet taxa showed a patchy distribution pattern. Plants have been encountered in the six major areas across the Desembarcadouro islet. In several of these areas B. patula shares the habitat with other wild beet taxa. Fig. 18 shows the preliminary results of the species‟ census and illustrates the relative frequency of wild beet taxa in each site.

Ecological/ environmental data Compared to the Ilhéu Chão the relief in the Desembarcadouro islet is more variable ranging from 0 to 104 m asl in the highest place. The mean annual temperature is higher than 18º C and the mean annual rainfall is less than 800 mm. The relative air humidity ranges between 60-70%. Clay is the prevailing soil type, the soil is rocky, poor in organic matter, of low soil moisture, the pH-value near to neutral and the salinity is high.

Figure 19. Census areas on islet Desembarcadouro

The Desembarcadouro islet can be roughly divided in three different soil units: Haplic calcisoils, Eutric accident soils, and Eutric rocky soils (FAO, 1988). B. patula occurs in the sites Bp-A1 to Bp-A3 which are characterized by the haplic calcisoil type. Within the sites Bp-A2 and Bp-A3 B. patula is

25 distributed continuously. The remaining B. patula patches were found on Eutric accident soil and Eutric rocky soil units, respectively (Fig. 19).

The Bp-A4 site showing the highest species diversity merges with Bp-A3 in the east side and with Bp- A5 in the west side. However, in the site Bp-A4 B. patula shows a fragmented distribution, maybe as a result of the relief and vegetation barriers. The Bp-A1 and Bp-A6 B. patula patches occur in the west and east end of the Desembarcadouro islet, respectively.

Table 12. Table presents the preliminary results of fieldwork in Desembarcadouro islet, with the number of specimens of the 3 Beta CWRs counted and sampled.

Species B. patula B. vul. subsp. maritima P. procumbens Location Census site Individuals counted and sampled Desembarcadouro A1 3 23 29 A2 19 63 38 A3 68 47 24 A4 223 30 38 A5 64 30 28 A6 38 - -

Genetic Under local conditions B. patula it is a biennial plant with stems reaching up to 30 cm, branching freely from the base, the branches spreading or ascending. The species is self-fertilising but outcrossing is easily possible (Letschert, 1993). The glomerules are composed of seven flowers on average. At seed maturity the seed balls of B. patula contain the highest number of seeds compared to all Beta species (Letschert, 1993). The phenotype of B. patula as expressed in the natural habitat is displayed in Fig. 20.

Figure 20. Phenotype of B. patula in the natural habitat

The genetic variability within the species has never been investigated with original material. Since the collection of B. patula seeds by Coons in the 1930s the material it has been regenerated at least several times and was exchanged in small seed quantities as research material between germplasm collections in Europe and the USA during the past decades. Letschert (1993) used 10 allozymes to investigate the genetic diversity within Beta section Beta species. At that time the only available ex situ accession of B. patula was IDBB6963 (parallel numbers are BGRC56782, BETA866, WB96) which was used to determine the infraspecific diversity. The genetic diversity (He) of B. patula turned out to be very low (He = 0.01) compared to B. vulgaris subsp. maritima (He = 0.28).

However, in general the genetic diversity between accessions of a self-fertilizing species is higher than the within accession diversity. Hence, the species B. patula as such may contain more genetic diversity than found by Letschert (1993) The low level of genetic diversity within IDBB6963 can be explained by genetic bottlenecks (low number of plants sampled during collecting mission) or low effective population sizes during seed regeneration causing genetic drift.

An investigation of the genetic diversity of the species was therefore initiated within the framework of the AEGRO project. The genetic diversity between plant groups sampled on Ilhéu Chão and area A1 to A6 on Ilhéu do Desembarcadouro was analysed using 22 SSR markers. For that purpose leaves of individual plants were sampled (see Tab. 11). A differentiation between the plant groups sampled on A1 to A6 was found. The plant group sampled on Ilhéu Chão was more distinct from the groups sampled on the larger islet. However, the first and second dimension of the principle component analysis explained 42.5% of the variation, only. The total number of alleles increased from the western

26 marginal sampling site A1 to A5 and slightly declined towards A6 in the eastern part. All plant groups contain private alleles. The expected heterozygosity was calculated for each marker and ranged between 0.12 to 0.83. The fixation index ranged from 0.11 to 0.53 indicating a deficiency of heterozygotes which underpins the assumption that B. patula is a self-compatible species. More details of this investigation will be published by Enders (2010).

27 Step 4: Selection of target sites Data on the spatial structure of genetic diversity of Beta / Patellifolia species will be generate by the national Portuguese project. The recommendation of candidate sites (Fig. 21 and 22) given in this study is based on the currently available knowledge.

Mainland Portugal

B. vulgaris subsp. maritima and B. macrocarpa

A) Aveiro

B) Fuseta

Figure 21. Shows the location and limits of protected areas in mainland Portugal and their coincidence with B. vulgaris subsp. maritima occurrences. Recommended locations for a genetic reserve: A) Aveiro and B) Fuseta.

28

Two locations within protected areas could be taken into consideration to represent genetic diversity of B. vulgaris subsp. maritima within a European wide set of genetic reserves. These two genetic reserves locations represent major climatic zones of Portugal.

The first one could be Aveiro coinciding with subclimate type 50a where approximately 500 individuals were observed by Frese et al. (1990). The area belongs to a Special Protection Zone mainly devoted to bird protection and is a Natura 2000 site. The region is strongly influenced by agriculture and other economic activies of a large human population.

The second one could be the salt winning area at Fuseta which homes B. vulgaris subsp. maritima and diploid B. macrocarpa which is considered threatened in Portugal and thus was included into the national priority list. Fuseta belongs to the Parque Natural da Ria Formosa, Corinne Code C23000010. The establishment of a genetic reserve there would allow the conservation and active management of two species at the same location.

Figure 22. shows the location of protected areas and occurrences of B. macrocarpa in mainland Portugal

Unlike B. vulgaris subsp. maritima the He value of all six occurrences of B. macrocarpa from Portugal and Southeast Spain investigated by Letschert (1993) proved to be zero. Fixed alleles within the populations and highly specific and highly threatened habitats underpin the need for a stronger protection of the species and management of the site creating the ecological niches B. macrocarpa requires for survival.

P. patellaris

This species was reported to occur at Cabo Espichel (Natura 2000 Code PTZPE0050, Special Protected Area EC Birds Directive) (Duarte, 2008). Since it seems to be the only distribution area of P. patellaris known for mainland Portugal the plants may be genetically different from the Spanish, the Madeiraean and Northwest African plants and should be surveyed. Before recommending the establishment a genetic reserve the site should be visited, a census made and samples taken for a comparative genetic analysis of this isolated plant group and those plants existing in Spain (mainland, Canary islands).

Archipelago of Madeira

B. patula, B. vulgaris subsp. maritima, P. procumbens

As the distribution area of B. patula is very small the selection of a genetic reserves site is straightforward. The Ilhéu do Desembarcadouro is much easier accessible than Ilhéu Chão, it belongs to a protected area, and homes two additional wild beet species. A higher diversity of SSR marker exists in the central / eastern part of the islet. Enders (2010) therefore suggested establishing a genetic reserve in that part of the islet. With respect to the very limited distribution area and the fact that private alleles occur within all sampled areas the delineation of a particular part of the islet may 29 not be necessary in particular if the increase of size does not result in increasing management costs. Ponta de São Lourenço belongs to the Natura 2000 Network (Code PTMAD0003). The recommended genetic reserve would be located in an area which is partially protected according to regional regulation No. 1408/2000, JORAM no. 85, 22 September 2000. The proposed designation of Ilhéu do Desembarcadouro as a genetic reserve for B. patula is based on practical considerations and may not be interpreted as a decision against Ilhéu Chão, where a unique fraction of the species‟ genetic diversity is located. As long as the information on the distribution pattern of genetic diversity within the Patellifolia species is very scarce, the site at Câmara de Lobos, Ponte dos Socorridos on the main island of Madeira is recommended as a candidate site. Madeira is the most northern and isolated part of the distribution area of B. procumbens. Hence, this site and Ilhéu do Desembarcadouro on the Archipelago may represent a specific and distinct fraction of genetic diversity.

30 Germany

Step 2: Selection of target taxa for the detailed national-level case study

In Germany only B. vulgaris subsp. maritima occurs. It can be assumed that the German sea beet plants form part of a larger group distributed in the western part of the Baltic Region (Denmark, southern Sweden).

The distribution of B. vulgaris subsp. maritima (BVM) has been described by Driessen et al. (2001). Additional data were retrieved from GBIF, EURISCO and GRIN, processed and uploaded to CWRIS AEGRO PLIS.

Step 2 a and b: Prioritized taxon list The compilation of a priority list is easily done (Tab. 13).

Table 13: Prioritized taxon list

Country Taxon Value as Threat according to national list National priority gene donor According to Ludwig et al. (2007) Germany BVM Yes No, extremely rare but not No particular protected responsibility

Step 3: Ecogeographic diversity analysis by species

Germany

Fig. 23 depicts maps showing the distribution of B. vulgaris subsp. maritima in Germany and adjacent areas. Although there are also spontaneous plants of this subspecies in inland areas, the main and natural distribution area is Helgoland, an island located in the North Sea, and isolated from the continent by 50 km distance, and the western shores of the Baltic Sea, where the species occurs more frequently and in several sites with a high number of individuals.

31

Figure 23: Distribution of B. vulgaris subsp. maritima in Germany and the adjacent areas along the Baltic Sea coast of Denmark and Sweden.

Figure 24: Habitat types of B. vulgaris subsp. maritima in Germany

32 Ecological/ environmental data

B. vulgaris subsp. maritima occurs in coastal shingle habitats in the North Sea (on the island Helgoland, EUNIS B2.2) and along the Baltic Sea coast on the island Fehmarn (more frequently in EUNIS B2.3) and the opposite coast of Schleswig-Holstein (EUNIS B2.2) (Fig. 24).

The climatic conditions of both parts of the distribution area are depicted in Fig. 25 for Helgoland and for the region of Ostschleswig-Holstein to which the island Fehmarn belongs to.

Figure 25. Climatic diagrams of Helgoland and Eutin (west of Fehmarn)

The annual average precipitation is identical, although more evenly distributed in the area of Ostschleswig-Holstein. The annual average temperature is 0.9 °C higher on Helgoland. During wintertime the average monthly temperature never falls below zero which explains why this frost susceptible wild beet species forms large, old and stable populations in Germany at sites such as the village of Lemkenhafen on Fehmarn.

The population on Helgoland grows on salty, stony clay soil within the spray water zone. Plant groups on Fehmarn and the adjacent mainland cost occur above the drift line in sandy clay soils between gravel and boulders (Fehmarn bridge, Puttgarden), on sandy soil (Großenbrode) or on clay soil rich in organic substance (Lemkenhafen).

Genetic

B. vulgaris subsp. maritima is a wind pollinated obligatory outbreeder. Cross fertilisation is controlled by three different systems: genetic male sterility, cytoplasmic male sterility and self-incompatibility. Genetic information is also exchanged between plant groups by seeds. The seed ball of the B. vulgaris subsp. maritima is composed of 3-5 seeds. About 50% of them still germinate even after 16 weeks of storage in sea water (Driessen et al., 2001). The extent of gene flow between plant group along the coast depends on several factors: landscape elements that may hinder the long distance dispersal of pollen by wind, the main wind direction during flowering, habitat type (cliff or drift line) (Raybould et al., 1997) and in particular sea currents (Fievet et al., 2007).

Information on the spatial structure of genetic diversity of B. vulgaris subsp. maritima in the German and Danish distribution area is provided by GMO sugar beet risk assessment studies (Driessen et al., 2001, Andersen et al., 2005). Driessen et al. (2001) used 75 RAPD fragment loci to describe the degree of polymorphism in five Danish and five German sea beet occurrences (such as GE-2 and DK-3, see below). They found a higher average polymorphism (20%) in the Danish material compared to 15% in the German plant groups. However, a one-way analysis of variance which included a third group as control showed that the differences in polymorphism were not significant between groups.

A close genetic relationship between Danish and German plant groups is further substantiated by Bartsch et al. (1999) who calculated the genetic distance of 22 European sea beet plant groups

33 based on thirteen polymorphic allozymes loci. Two of their entries were of identical geographic origin as GE-2 and DK-3. These two entries described by Bartsch et al. (1999) clustered closely while the plant group from Helgoland clustered closely with an Irish accession. Strikingly, the collectors of accession number BGRC62760 noted in 1993 that the plants on Helgoland look like the Irish (IDBB, http://idbb.bafz.de/CCDB_PHP/idbb/ visited on 13 August 2009). If the plant group occurring on Helgoland contains a fraction of the genetic diversity present in British and Irish occurrences, then the establishment of a genetic reserve on Helgoland may not be necessary.

Andersen et al. (2005) sampled 12 plant groups in Denmark and 2 in Sweden and compared them with accessions from Italy, France, Ireland and The Netherlands. Their entry DK3 is not identical with DK-3 described by (Driessen et al., 2001) but it was possibly sampled at Kalundborg Fjord, the origin of Rizomania resistance gene widely used in sugar beet breeding. Andersen et al. (2005) described the genetic diversity of these 18 entries with eight SSR markers. The analysis of molecular variance showed that 69.01% of the total variance can be explained by the within populations variance, 20.14% by differences between the five geographic groups, and only 10.85% by differences between groups within a region which means, that the Baltic sea beet plant groups share a large fraction of genetic diversity in common.

Frese revisited occurrences on the island of Fehmarn (Germany) and Denmark in 2008, georeferenced individual plants within 5 sampling areas and collected leaf samples of about 48 individuals per site. G. Poulsen (NordGen) sampled in a similar way two additional sites in Denmark, however, without georeferencing the individuals. Similar to the genetic analysis of B. patula the material was analysed by Enders (2010). The principal component analysis yielded two plant groups (Lolland/Vesternaes, Sjaelland/Kalundborg Fjord) which were very distinct from a third large group. The latter was composed of plants sampled on the Danish and German remaining 5 sites, amongst which was the site Lemkenhafen on Fehmarn.

Step 4: Selection of target sites The establishment of a genetic reserve for the subspecies B. vulgaris subsp. maritima should be recommended in due consideration of its wide distribution along the European Atlantic and Mediterranean coasts. In the Baltic Sea area it may even spread as indicated by the many recent sightings of very large, old and stable plants groups as reported by Driessen et al. (1997) and Andersen et al. (2005).

A recommendation must also take into account the immense value of the sea beet as a genetic resource for sugar beet breeding. Surveys in 2008 and 2009 (Frese, unpublished) of populations in Denmark, Germany and France indicate that the sea beet survives very well if the habitat remains untouched and unchanged. It could be argued that the operation of genetic reserves will not be expensive. The active management would only require a census monitoring at rather large time intervals (5-10 years). Although the species is not threatened within in Germany and the adjacent Danish region, the region itself represents a unique part of the whole distribution area. A few locations could be designated as genetic reserve to make sure that the specific fraction of genetic diversity occurring in the region continues to evolve.

A very unique location is the FFH site DE1813391 “Helgoland mit Helgoländer Felssockel” where the sea beet forms part of the perennial vegetation of the gravel beaches (NATURA 2000-Code: 1220) (Fig. 26). Biological monitoring is being implemented in this area (last update: 2009-07-21), however, it does not include the Code 1220 habitat where the sea beet grows. In 1993 the population had an effective population size of 100 seed parent plants so that at least a census baseline is there (IDBB, http://idbb.bafz.de/CCDB_PHP/idbb/ , visited on 13 August 2009).

34

Figure 26. The green arrow points to the growing site of B. vulgaris subsp. maritima, the white arrow to the only occurrence of wild Brassica oleracea in Germany. Helgoland could be designated as a two species genetic reserve location.

In addition, two sites in the Baltic Sea region are suggested as candidate site. The Kalundborg Fjord location can be recommended for historical, pragmatic and biological reasons. First, collections made in the 1950s provided Rhizomania resistance genes in use today, second the area is not disturbed by tourism and third the population is very large and stable.

Andersen et al. (2005) described a larger plant group north of Bagenkop on the southern tip of the island Langeland. Up to 1000 plants are growing in this area indicating a stable population. An equally stable group of plants occurs at the opposite coast at Lemkenhafen in Germany. The bay of Lemkenhafen (Fig. 27) as well as the small coast strip where the sea beet occurs is part of FFH DE 1532-391 “Küstenstreifen West- und Nordfehmarn”. If genetic resources of the sea beet is to be actively managed in a broader European context, the German contribution could consist in the establishment of two genetic reserves for Beta, the first located on Helgoland (FFH DE1813391) and the second in the bay

Figure. 27. Bay of Lemkenhafen. Green area = growing site of B. vulgaris subsp. maritima of Lemkenhafen (FFH DE 1532-391). This is the conclusion of the German national level case study on Beta.

Sites recommended in part A and B are compiled in Appendix 1.

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39