Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 Contents lists available at ScienceDirect Perspectives in Plant Ecology, Evolution and Systematics journal homepage: www.elsevier.de/ppees Biological Flora of Central Europe Biological flora of Central Europe: Leucojum aestivum L. Gilberto Parolo a,∗ , Thomas Abeli a , Graziano Rossi a , Giuseppina Dowgiallo b , Diethart Matthies c a Department of Earth and Environmental Sciences, University of Pavia, Via S. Epifanio 14, I-27100 Pavia, Italy Department of Biologia Vegetale, University of Rome, La Sapienza, Piazzale Aldo Moro 1, I-00185 Roma, Italy c Department of Ecology, Faculty of Biology, Philipps-University, Marburg D-35032, Germany b a r t i c l e i n f o Article history: Received 14 September 2010 Received in revised form 5 April 2011 Accepted 18 May 2011 Keywords: Conservation status Density effects Ecological niche Population size Reproductive biology Seed germination a b s t r a c t Leucojum aestivum L. (Amaryllidaceae) is a polycarpic C-S-European/W-Asiatic geophyte. It is a threatened wetland species and is protected in several European countries, as a consequence of the destruction or alteration of its habitats across Europe and the harvesting of its bulbs for medical purposes (alkaloids). This paper deals with the taxonomic status, morphology, distribution, ecology and population biology of this species, with special emphasis on habitat requirements, reproductive biology, and seed germination. A detailed study in N-Italy found that L. aestivum grows on alluvial soils with high nitrogen levels. The mean size of the plants increased with the water content of the soil. Similarly, within the habitats, the water and nitrogen contents of the soil were higher in plots with L. aestivum than in those without the species. Seed set of the plants was not influenced by the size of a population, but strongly increased with the density of flowering plants. This was due to a decrease in the proportion of unfertilised eggs, indicating pollen limitation of reproduction in low-density populations. Germination tests revealed that the optimal germination temperature is between 20 ◦ C and 25 ◦ C. © 2011 Perspectives in Plant Ecology, Evolution and Systematics. Published by Elsevier GmbH. All rights reserved. Taxonomy and morphology Leucojum aestivum L. Sp. Pl. 1: 289 (1753); Gen. Pl. ed. 5: 140 (1754). (Amaryllidaceae) – Sommer-Knotenblume, Summer Snowflake, Snowflake Lily, Loddon Lily, Campanellino estivo, Campanilla de Primavera, Copo de nieve. L. aestivum subsp. aestivum =Nivaria monodelphia Medic. 1790. Act. Acad. Theod. Palat. Phys. VI: 422. =Polyanthemum aestivale Bubani 1902. Fl. Pyr. 4: 155. (nom. illeg.) L. aestivum subsp. pulchellum (Salisb.) Briq. 1910 Prodr. Fl. Corse 1: 323. =L. pulchellum Salisb. 1807. Parad. Lond. t 74. =L. hernandezii Cambess. 1827. Mem. Mus. Par. XIV: 315. =L. hernandezianum Schultes & Schultes fil. in Roemer & Schultes 1829, Syst. Veg. ed. 15, 7(2): 784. Taxonomy Molecular studies have suggested that the genus Leucojum is closely related to Galanthus L. (Meerow et al., 1999; Ito et al., 1999; Lledó et al., 2004; Table 1). This hypothesis was first formulated by Stern (1956), who located the origin of the common ancestor of the ∗ Corresponding author at: Dep. Earth and Environmental Sciences, University of Pavia, Via S. Epifanio 14, I-27100 Pavia, Italy. Tel.: +39 0382 984854; fax: +39 0382 34240. E-mail address: gilberto.parolo@unipv.it (G. Parolo). two genera in the Mediterranean area or in Central Europe. Meerow et al. (2006) suggested that North Africa and the Iberian Peninsula are the most likely areas of origin of this clade. In contrast, based on cytological studies, D’Amato and Bianchi (1999) had found no evidence for a common origin of the two genera. The genus Leucojum consists of 11 species, belonging to four groups or subgenera (Table 2). L. aestivum is the only species of the subgenus Aerosperma (Baker, 1888; Contandriopoulos, 1962). Like the subgenus Leucojum that includes only L. vernum L., the subgenus Aerosperma is characterized by hollow scapes, wide leaves and clavate styles. Taxa from both subgenera are diploids with a base chromosome number of n = 11 (Lledó et al., 2004). Subgenus Ruminia (Parl.) Baker includes three narrow endemics: (1) L. valentinum Pau from eastern Spain, whose records from Greece in the literature are considered to be erroneous (Bareka et al., 2006; Jordan-Pla et al., 2009); (2) L. nicaeense Ardoino from the Maritime Alps of southern France and NW-Italy (Diadema et al., 2004; Conti et al., 2005), and (3) Leucojum fabrei Quézel and Girerd (Diadema et al., 2004) from Mount Ventoux in France. The subgenus Ruminia is characterized by solid scapes, narrower leaves and filiform styles (Lledó et al., 2004). The largest of the four subgenera is Acis (Salisb.) Baker (Lledó et al., 2004), which includes two Atlantic species, L. trichophyllum Schousb., occurring from Portugal to the Atlantic coast of Morocco and North Africa, and L. autumnale L., with a distribution overlapping that of the previous species and reaching also Sicily and Sardinia (Conti et al., 2005). There are also three narrow 1433-8319/$ – see front matter © 2011 Perspectives in Plant Ecology, Evolution and Systematics. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ppees.2011.05.004 320 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 Table 1 Systematic position of Galanthus, Leucojum and related genera (after Lledó et al., 2004). the phenological isolation between submediterranean/continental populations. Following Traub (1970) Following Müller-Doblies and Müller-Doblies (1978) Karyology Intrafamily Amaryllidoidinae Tribe Narcisseae Stembergia Narcissus Tribe Galantheae Lapiedra Hannonia Leucojum Galanthus Intrafamily Pancratioidinae Tribe Pancratieae Pancratium Vagaria Tribe Narcisseae Subtribe Narcissineae Stembergia Narcissus Subtribe Galanthinae Leucojum Galanthus The chromosome number of L. aestivum is 2n = 22 (Barros-Neves, 1939). The same number was found by other authors for material of different origin, e.g. Chiappini and Scrugli (1970), D’Amato and Bianchi (1999), Senel et al. (2002), and Bareka et al. (2003). This chromosome number is considered plesiomorphic for the Amaryllidaceae family, due to its occurrence in many tribes of the family (Meerow et al., 1999). However, other chromosome numbers have also been reported (Magulaev, 1986; Özhatay, 2002: 2n = 24; Dobeš et al., 1997: 2n = 21, 22, 23, 24). L. aestivum has one long, paired metacentric chromosome, one pair of medium subtelocentric chromosomes and ten pairs of acrocentric chromosomes, ranging in size from 3.1 to 16.6 ␮m (Bareka et al., 2003). The mean base karyotype length is 89.65 ␮m ± 2.9 and the symmetry index 32.47 (D’Amato and Bianchi, 1999). Tribe Pancratieae Subtribe Pancratiinae Pancratium Vagaria Hannonia Subtribe Lapiedrinae Lapiedra Morphology and anatomy endemics: one from Corsica (L. longifolium J. Gay ex Salisb.), one from Sardinia and Corsica (L. roseum Mart.), and a third from North Africa (L. tingitanum Baker) (Lledó et al., 2004; Conti et al., 2005). A fourth species, endemic to W-Greece and S-Albania, is L. ionicum Kit Tan, Mullaj, Sfikas and Strid, described by Tan et al. (2004). Recently, Bareka et al. (2006) and Meerow et al. (2006) considered the subgenus Acis to be a separate genus. Characters distinguishing the four subgenera are summarized in Table 2. Two subspecies of L. aestivum are known: L. aestivum L. subsp. aestivum and L. aestivum L. subsp. pulchellum (Salisb.) Briq. L. aestivum subsp. aestivum is a Linnaean species (Linnaeus, 1753), whose holotype has not been designated yet (Jarvis, 2010). Loci classici are “Pannonia, Hetruria, Monspelii, Austria” (Linnaeus, 1753). L. aestivum subsp. pulchellum was described by Salisbury (1807–1808), probably based on a plant growing in a botanical garden in London, since the material figured in its book Paradisus Londinensis came from gardens in the Metropolis. However, to our best knowledge, no further information is available about the typus specimen and the locus classicus (Fred Rumsey, Natural History Museum London, Odile Weber and Paul Wilkin, RBG Kew pers. comm.). Most authors recognize the two subspecies as valid taxa (Tutin et al., 1980; Conti et al., 2005), but some have considered the subsp. pulchellum only as a Mediterranean variety of L. aestivum, and as part of the variability of L. aestivum subsp. aestivum (Chiappini, 1964; Chiappini and Scrugli, 1970). We agree with Tutin et al. (1980) and Conti et al. (2005) in considering these two subspecies as valid taxa, on the basis of their different, but adjacent geographic distribution, and phenological and morphological features (see morphology and anatomy). Since L. aestivum subsp. pulchellum flowers 2–3 weeks earlier than the subsp. aestivum, differentiation of the two subspecies may have started as a consequence of L. aestivum is a perennial bulbous geophyte (Fig. 1). The bulb is sub-spherical (up to 6 cm in diameter), with a brown tunic (Tutin et al., 1980), which helps the plant to survive in the summer dry period; it contains the basal plate, fleshy scales, 1–4 shoots, and sometimes lateral buds. Adult bulbs were found to be 10.7 ± 1.2 cm below the soil surface (n = 10, Pavia, N-Italy). The bulb shows a sympodial branching system, each unit of which is composed of 6–8 foliage leaves, and a lingulate scale, and terminates in an inflorescence (Mori et al., 1991). Rarely, we observed anomalous bulbs developing one above the other and linked by a pulpy protuberance 4–6.5 cm long (see Gay, 1859). This appears to be a reaction to a covering of the bulbs by sediments; in this case the bulb axis elongates to bring it to the appropriate level. The root system is not ramified and roots start to grow usually at the end of the summer/beginning of autumn. Contractile roots, that can pull the bulb deeper, are present only in subadult plants (about a third of the roots); contractile roots are slightly thicker than normal roots (about 2.5 mm thick). In N-Italy, 3-year old plants were found to have 12 ± 4 roots (n = 12); the length of the root system of adult plants was 14.5 ± 2.8 cm (n = 16). The radius of the root system was 9.6 ± 2.9 cm. The leaves are broadly linear, amplexicaule with a lamina 10–110 cm long and 5–20 mm wide during the vegetation period; the bases of foliage leaves, which encircle the axis and gradually enlarge, serve as a food storage organ (Mori et al., 1991). The scape is stout, hollow, compressed with two serrulated hyaline margins. The inflorescence is a single helicoidal cyme subtended by a spathe formed of a single two-keeled leaf, with (1) 2–8 (11) pendulous flowers. Each plant can produce up to 3 (4) flowering scapes (or peduncles), but more commonly only one. Spatha bracts Table 2 Characters distinguishing the four subgenera of Leucojum (modified from Lledó et al., 2004). Subgenus Scape Longest floral pedicel Perianth Outer perianth segment Gynoecium Length of seed Seed characters Base chromosome number Leucojum Aerosperma Acis Hollow Hollow Solid Equal or longer than spathe Equal or longer than spathe Shorter than spathe With spots With spots No spots Callose-thickened Callose-thickened Apiculate or acute 5–7 mm 5–7 mm 1–3 mm Pale Black; no appendage Greenish; no appendage n = 11 n = 11 n = 7, 8 Ruminia Solid Shorter than spathe No spots Apiculate or acute Style clavate Style clavate Style filiform; unlobed epigynousdisc Style filiform; unlobed epigynous disc 1–3 mm Greenish with appendage n=9 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 321 A layer of papillous cells forms the tepal epidermis. Just below it other cells, containing needle-shaped chloroplasts, occupy the green parts of tepals. Chlorophyll concentration in the green parts of tepals is fivefold lower than in the leaves (Ščepánková and Hudák, 2004). L. aestivum is morphologically similar to L. vernum from which it differs by its larger size, an inflorescence with more than one flower, the dark coloured seeds lacking appendages, and the adaptation to water dispersal (Lledó et al., 2004), instead of myrmecochory (Servigne, 2008). Furthermore, L. aestivum flowers generally a month later than L. vernum. The differential characters of the two subspecies are: L. aestivum subsp. pulchellum has leaves 5–12 mm broad, scape margin neither denticulate, nor hyaline, bracts 30–50 × 4–6 (–7) mm, flowers 1–5, tepals 8–15 mm long (Salisbury, 1807–1808); L. aestivum subsp. aestivum has leaves 7–20 mm broad, bracts 30–50 × 7–11 mm, flowers (1-)3-8(11), tepals 13–22 mm long. Distribution and habitat requirements Geographical distribution Fig. 1. Morphology and anatomy of an adult plant of Leucojum aestivum subsp. aestivum (longitudinal section). 2, fused entirely in one side, 3–5 cm long, 7–11 mm wide. Pedicels are 2–7 cm long and the longest can exceed the spatha. The perianth is formed by 3 + 3 tepals (2 whirls), 13–22 mm long, with a green spot just below the apex. The style is slightly clavate and longer than the 3 + 3 orange stamens (Tutin et al., 1980); the anthers dehisce by terminal pores. The gynoecium is 3–14 mm long and contains 10–40 bitegmic crassinucleate ovules, supported by a marginalcentral placenta (Ekici and Dane, 2008). The fruit is a sub-spherical, fleshy capsule 2–4 cm long and 1–2 cm wide. The seeds are large (5–7 mm), black, spherical, without strophiole and with a spongy testa which allows them to float (Tutin et al., 1980; Pignatti, 1982). The moisture content of seeds has been determined as 33% (Çiçek et al., 2007) and their mass as 94 mg (Çiçek et al., 2007) and 94.3 mg (n = 405 seeds, own measurement). The anatomy of leaves and tepals has been studied by Ščepánková and Hudák (2004). The epidermis is covered by a prominent cuticule and the mesophyll cells form a homogeneous parenchymatic tissue with many intercellular spaces a typical feature of equifacial leaves. Leaves are amphistomatic, with anomocytic stomata. The mesophyll cells are isodiametric with chloroplasts along the cell walls. Leaves show some prominent structures that are large central cavities filled with a mucose substance and separated by vascular bundles. Leaf chloroplasts have an irregular, amoeboid shape and typical grana and stroma lamellae. A detailed description of the chloroplast of L. aestivum can be found in Ščepánková and Hudák (2004). Most of the Leucojum species are found in the western Mediterranean region. L. aestivum is a C-S-European/W-Asiatic species whose distribution in the north reaches Ireland, Czechia and Slovakia, and in the east Turkey, the Crimea and Iran (Tutin et al., 1980; Crellin, 2005; Fig. 2). The species occurs from sea level up to 350 (1000) m (Pignatti, 1982; Çiçek et al., 2007). From the eastern to the western limit of its range, L. aestivum subsp. aestivum occurs south of the Caspian Sea, in northern Iran (Mazhari, 2004), a disjunction which shows that the plant distribution has been formerly broader; in the Caucasus in Azerbaijan (Wendelbo, 1970) and in Georgia (Denk et al., 2001); in Turkey (Kutbay and Kilinç, 1993; Ekici and Dane, 2008); in SW-Russia and the Ukraine in Crimea (Marushevsky, 2003; Kohut et al., 2007), and along the coast of the Black Sea (Tutin et al., 1980); in Bulgaria in the Rhodope Mountains and along the Danube River (Stanilova et al., 1994; Bondev, 1995; Gussev et al., 2003); in Hungary (Makra and Zalatnai, 2006); in Romania and Moldova (Sârbu, 2007; Mardari et al., 2009); in mainland Greece (Bergmeier, 1988; Arne Strid pers. comm.; Schuler, 2007), Peloponnesos (Arne Strid pers. comm.) and in some of the Greek islands (Bareka et al., 2003; Schuler, 2007; Vladimirov et al., 2009); in Albania (Pinna et al., 2007), in Serbia (Jovanović et al., 2009), Slovenia (Cater et al., 2001) and Croatia (Čarni et al., 2004); in Slovakia and the Czech Republic (Somsak, 1992; Uherčíková, 1998); in Italy in the plain of the river Po, and in Tuscany (Poldini, 1997; Conti et al., 2005; Parolo and Rossi, 2008); in Austria (Fischer et al., 2008); in France (Molinier and Tallon, 1970; Guinochet and de Vilmorin, 1978; Aizpuru et al., 2001; SILENE, 2006–2010); in Switzerland (Hess et al., 1967; Welten and Sutter, 1982; Moser, 1999; Lauber and Wagner, 2000). The rarer L. aestivum subsp. pulchellum is restricted to the Western Mediterranean region (Tutin et al., 1980). It grows in Italy, in Sardinia (Chiappini and Scrugli, 1970; Pignatti, 1982; Conti et al., 2005); in Corsica and in mainland France (Médail et al., 1994; Moser, 1999; Frédéric Médail pers. comm.); in Spain on the Balearic Islands of Mallorca and Menorca (Guinea Lopez and Ceballos Jimenez, 1974; Bolòs and Vigo, 2001). The subsp. aestivum also occurs, as an introduced plant, in Belgium (Lambinon et al., 2004), in the Netherlands (Tutin et al., 1980), and in Germany (Buxbaum, 1934; Oberdorfer, 2001). Populations of both L. aestivum subsp. aestivum and subsp. pulchellum are known from the United Kingdom, but most of the populations are derived from introduced plants (Stace, 2010). L. aestivum subsp. aestivum is native only along the River Thames and its tributaries (Farrell, 1979; Wigginton, 1999; Fred Rumsey, pers. comm.), where 322 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 Fig. 2. Distribution of Leucojum aestivum subsp. aestivum and L. aestivum subsp. pulchellum based on floristic literature and floristic databases (provided by Dr. E. Welk). it reaches the northern limit of its natural range (Preston, 2007). The subsp. pulchellum has been in cultivation in the U.K. for several centuries and much of the material now found in the wild in Britain belong to this subspecies, escaped from cultivation or has been deliberately planted (Fred Rumsey, pers. comm.). In Ireland L. aestivum subsp. aestivum is considered to be native at a few sites. However, also in Ireland many populations of both subspecies are the result of past introductions (Farrell, 1979; Farrell, 1982). The species is also naturalized or cultivated outside of its natural range as an ornamental plant, e.g. in the USA (Straley and Utech, 2002), Australia (George, 1987; Rippey et al., 2007), New Zealand (Healy and Edgar, 1980), Japan (Mori et al., 1991a) and South America (Hurrel and Delucchi, 2007). Habitats In the valley of the river Po (N-Italy), where most of the Italian populations of L. aestivum subsp. aestivum grow, winters are relatively short, but foggy, spring and autumn are well marked, while summers are generally hot and humid, but with little precipitation. In Britain L. aestivum grows in regions where the mean January temperature is c. 4.3 ◦ C, the mean July temperature is c. 15.9 ◦ C, and mean annual precipitation reaches 811 mm (Hill et al., 2004). In its native distribution area L. aestivum subsp. aestivum occupies alluvial habitats near rivers, lakes or on the banks of canals, and humid, often periodically flooded sites from 0 to 350 (1000) m a.s.l., which are shaded, semi-shaded or in full light (Fitter, 1978; Ellenberg, 1988; Oberdorfer, 2001; Zagorska et al., 1997; Čarni et al., 2004). The soil of the habitats is usually sedimentary, deep and compact, consisting of clay or loam, with a very fine texture and hydrophilic character (Athanasiadis and Drossos, 1992; Stancic, 2005). It is rich in nitrate and humus and poor in carbonates, with a moderately acid to slightly basic pH. L. aestivum occurs also far from water bodies, but only where a sufficient amount of water in the soil is available during the growing season from February to the end of May; it never occurs in ruderal habitats as an early successional plant (Moser, 1999). In its habitats, the plant generally occupies microdepressions, where soil moisture is higher (Stancic, 2005). Nevertheless, during summer the plant can be exposed to drought, as a consequence of the lowering of the water level. A detailed study of 26 populations of L. aestivum subsp. aestivum in N-Italy found that the species was growing on alluvial soils, classified as Fluvisol (FAO, 1998). The soil characteristics were very variable due to the highly variable nature and composition of the fluvial deposits (Table 3). The mean size of plants in populations, measured as maximum leaf length, increased with the water content of the soil (r = 0.52, F1,24 = 9.0, p < 0.01). At most sites silt was the predominant soil fraction, but some sites had finer-textured topsoils with a high proportion of clay (>40%), mainly associated with wetland vegetation such as sedge and reed communities. The variable composition of the sediments was also reflected in a wide range of pH values (from acidic to nearly neutral) and of carbonate contents (Table 3). The organic carbon contents and total N levels were high or very high at all sites with a few exceptions and C/N ratios were rather low (mostly <10), indicating active mineralization processes (Duchaufour, 1995). Values of available P (P2 O5 ) were usually moderate to high (range 10–50 mg/kg); but at a few sites very high P-levels were found, most likely due to crop fertil- Table 3 Characteristics of the soil at 26 sites with Leucojum aestivum subsp. aestivum in N-Italy. Soil samples were dried at 105 ◦ C for 24 h and sifted through a sieve of 2 mm mesh width. Physical–chemical analyses were performed following the MIPAF (2000) standard protocol: pH was measured in a 1:2.5 soil/water suspension using a glass electrode; organic carbon (C) by Walkley–Black wet combustion; organic matter content was assessed as organic carbon times 1.726; total nitrogen (N) by Kjeldahl digestion; total calcium carbonate (CaCO3 ) using a volumetric calcimeter; particle-size distribution by the hydrometer method after a pre-treatment with H2 O2 to eliminate the organic matter. Available phosphorous (P2 O5 ) was measured colorimetrically after extraction with sodium bicarbonate (samples with pH >6.5) or ammonium fluoride (samples with pH <6.5). Variable Mean SD Range Sand (%) Silt (%) Clay (%) pH CaCO3 (%) C (%) Organic matter (%) C/N-ratio N (%) P2 O5 (mg/kg) Water content (%) 17.8 60.3 21.9 6.6 4.4 6.4 11.0 9.9 0.7 38.2 46.7 19.5 18.0 13.8 1.0 6.4 3.2 5.5 1.7 0.3 38.2 17 1.3–64.4 29.0–87.2 5.2–64.8 4.1–7.8 0–19.8 1.5–13.5 2.6–23.2 6.2–15.2 0.2–1.5 12.7–172.1 10.0–79.0 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 isation. These results are in broad agreement with the assessment of the realised ecological niche of the species by Landolt (1977), who classified L. aestivum as an indicator species for temporarily moist, nutrient-rich soils with a pH of 5.5–8, and for half-shaded conditions. In Central Europe the species is also an indicator of the warmest habitats, which are wintermild with little risk of late frosts (Landolt, 1977). Indicator values by Ellenberg et al., 2001 (indexes vary from 1 to 9 or 12 for F index) are: L (light) = 7, T (temperature) = 8, K (continentality) = 4, F (humidity) = 9, R (soil reaction) = 7, N (nutrients) = 8 and by Landolt (1977, indexes vary from 1 to 5) are: F (humidity) = 4, R (soil reaction) = 3, N (nutrients) = 4, H (soil humus content) = 4, D (soil dispersion) = 5, L (light) = 3, T (temperature) = 5, C (continentality) = 2. Very little is known about the habitats in which L. aestivum subsp. pulchellum grows. It occurs in regions with a mediterranean climate, characterized by warm to hot, dry summers and mild, wet winters (Cowling et al., 1996), and as an introduced plant also in regions with a temperate oceanic bioclimate (Rivas-Martinez and Rivas-Sáenz, 2009). According to Guinea Lopez and Ceballos Jimenez (1974) the species grows in mediterranean wet grasslands and on river banks. In SE-France it occurs at altitudes of 0–100 m and in Corsica of 0–300 m (Médail and Verlaque, 1997). In Mallorca it grows in mountain areas in Mediterranean Oak forests, shrub vegetation of gravel beds of seasonal streams and in tall wet grasslands. Plant communities L. aestivum grows in wetland plant communities, such as wet meadows, forests and scrubs in the lowland river floodplains and valleys (Ellenberg and Klötzli, 1972; Horvat et al., 1974; Tutin et al., 1980; Ellenberg, 1988; Somsak, 1992; Athanasiadis and Drossos, 1992; Mucina et al., 1993a,b; Grabherr and Mucina, 1993; Oberdorfer, 2001; Kutbay et al., 1998; Moser, 1999; Cater et al., 2001; Lazowski, 2001; Leonardi and Rossi, 2001; Makra and Zalatnai, 2006; Čejka et al., 2008; Parolo and Rossi, 2008; Onyshchenko, 2009). Table 4 gives a list of syntaxa in which L. aestivum occurs. The anthropogenic herbaceous plant communities in which the plant grows often replace former woods, which were adapted to inundations and seasonally high ground-water levels. Bergmeier (1988) suggested that in Greece L. aestivum is a relict species of former woods. Extensive vegetation surveys in 116 small random plots (2 m × 3 m) in 26 populations of L. aestivum subsp. aestivum in the valley of the river Po in N-Italy revealed the following: In speciespoor reed communities (Phragmition), L. aestivum subsp. aestivum can be common and sometimes dominant. It flowers before the main development of the reeds, thus avoiding the strong competition of Phragmites australis. In sedge communities (Magnocaricion elatae) L. aestivum is commonly associated with Carex elata, Carex acutiformis, Carex riparia, Carex hirta, Lysimachia vulgaris, Lythrum salicaria, Phalaris arundinacea. In wet meadows (Molinietalia) L. aestivum is often associated with Scirpus sylvaticus, Lotus pedunculatus, Caltha palustris, Myosotis palustris, Mentha longifolia, L. vulgaris, Filipendula ulmaria, Poa trivialis, Valeriana officinalis, Allium angulosum, Gratiola officinalis, Alopecurus utriculatus, Alopecurus pratensis, Ranunculus repens, Carex otrubae, C. riparia, and Poa sylvicola. In woods of Alnus glutinosa (Alnion glutinosae), L. aestivum can be dominant in the herb layer, where it is frequently associated with Thelypteris palustris, Humulus lupulus, Iris pseudacorus, Carex elata, C. acutiformis, Galium palustre, and Lysimachia vulgaris; in the shrub layer common species are Salix cinerea, Viburnum opalus, and Frangula alnus. In woods of Salix alba (Salicion albae) L. aestivum occurs scattered and is commonly associated with C. elata, C. acutiformis, Equisetum telmateja, Phalaris arundinacea, G. palustre, G. aparine, 323 Table 4 The main plant communities in which L. aestivum grows. 1. Phragmition communis Koch 1926 [reeds in slow-mowing water] 2. Magnocaricion elatae Koch 1926 [tall sedge swamps] 2.1 Phalaridetum arundinaceae Libbert 31 (Balkans: Horvat et al., 1974) 2.2 Caricetum vulpino-ripariae (Balkans: Horvat et al., 1974) 2.2 Eleochari-Caricetum nutantis (Balkans: Horvat et al., 1974) 2.4 Caricetum gracilis-vulpinae (Horvat et al., 1974) 2.5 Cyperetum longi caricetosum acutiformis (ass. char., Horvat et al., 1974) 3. Cirsio brachycephali-Bolboschoenion (Passarge, 1978) Mucina in Bal.-Tul. et al. 1993 3.1 Bolboschoenetum maritimi Eggler 1933 (Balkans: Horvat et al., 1974) 4. Calthion R. Tx. 1937 em. Bal.-Tul. 1978 [C-European manured moist meadows] 5. Cnidion Bal.-Tul. 1966 [subcontinental Cnidium meadows] 6. Molinion Koch 1926 (all. char., Horvat et al., 1974) [Molinia litter meadows] 6.1 Carex gracilis-Poa palustris plant community (Horvat et al., 1974) 6.2 Ventenato-Trifolietum pallidi (Horvat et al., 1974) 7. Deschampsion Horvat 1930 (all. char., Horvat et al., 1974) [SE-European manured moist meadows] 7.1 Deschampsietum cespitosae (Horvat et al., 1974) 7.2 Oenantho silaifoliae-Alopecuretum pratensis (Horvat et al., 1974) 8. Arrhenatherion Koch 1926 [Oatgrass meadows] 8.1 Arrhenatheretum elatioris holcetosum lanati (N-Croatia: Horvat et al., 1974) 8.2 Bromo-Cynosuretum cristati (NW-Bosnia: Horvat et al., 1974) 9. Trifolion resupinati K. Micevski 1957 (SE Europe: Horvat et al., 1974) [submediterranean meadows with annual Trifolium species] 10. Convolvuletalia sepium R. Tx. 1950 em. Mucina 1992 [semi-shaded nitrophilous riparianand lacustrine megaforb or climbing hygrophilous communities] 11. Alnion glutinosae Malcuit 1929 [Alder swamp woods] 11.1 Carici elongatae-Alnetum glutinosae var. Leucojum aestivum (Slovenia: Cater et al., 2001; Horvat et al., 1974). 12. Salicion albae Soó 1930 [Willow communities of lowland flood plains] 12.1 Salici-Populetum nigrae (ass. char., Danubian floodplain forests: Čejka et al., 2008; Serbia: Horvat et al., 1974) 12.2 Salicetum albae-fragilis (ass. char., Hungary: Makra and Zalatnai, 2006) 12.3 Salicetum albo-triandrae (Serbia: Horvat et al., 1974) 12.4 Leucojo-Fraxinetum angustifoliae (ass. char., Horvat et al., 1974) 13. Carpinion betuli Issler 1931 [mixed Hornbeam woods] 14. Alnion incanae Pawlowski in Pawlowski et Wallisch 1928 [Alder and broadleaved woodsof flood plains] 14.1 Fraxino pannonicae-Ulmetum (Ukraine: Onyschenko et al., 2009) 14.2 Fraxino angustifoliae-Alnetum glutinosae (Austria: Lazowski, 2001) 14.3 Leucojo-Fraxinetum parvifoliae (Greece: Athanasiadis and Drossos, 1992) 14.4 Querco-Ulmetum (Europe: Horvat et al., 1974) 14.5 Pruno-Fraxinetum (Horvat et al., 1974) 15. Populetalia albae Br.-Bl. 1931 [submediterranean White Poplar woods of flood plains] 16. Alno-Quercion roboris Horvat 1950 [Oak-Ash forests of flood plains] 16.1 Euphorbio palustris-Crataegetum nigrae leucojetosum aestivi (Croatia: Čarni et al., 2004) 16.2 Genisto-Quercetum roboris (ass. char., Horvat et al., 1974) 17. Ostryo-Carpinion orientalis Horvat 1950 (SE Europe: Horvat et al., 1974) [mixed deciduous and evergreen forests] 18. Quercion ilicis Br.-Bl. (1931) 1936 [Mediterranean Oak forests] 18.1 Cyclamini-Quercetum ilicis Alzinar. O.Bolós et R. Mol. 1908 (Mallorca). 19. Nerion oleandri Br.-Bl. et Bolos 1956 [Mediterranean shrub vegetation of gravel beds of seasonal streams] 20. Molinio-Holoschenion Br.-Bl. ex Tchou 1948 [Mediterranean tall humid grasslands] Iris pseudacorus, L. salicaria, Rubus caesius, and Urtica dioica. In elmoak woods (Carpinion betuli), L. aestivum is not abundant, probably because in these communities the water-table is not high enough. L. aestivum frequently occurs in non-native Amorpha fruticosa shrub communities, or in nitrophilous communities along river banks (Convolvuletalia sepium R. Tx. 1950 em. Mucina 1993a) with Rubus caesius, Urtica doica, Solidago gigantea, Phalaris arundinacea, Poa trivialis, and R. repens; it can also rarely be found in woods of Populus alba, and in plantations of Platanus hybrida. 324 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 According to the European “Habitats Directive” 92/43/ECC, in Sites of Community Importance L. aestivum can occur in the following habitats of the annex I of the directive (European Commision, 2007): 6410 Molinia meadows on calcareous, peaty or clay-silt-rich soils (Molinion caeruleae); 6430 Hydrophilous tall herb fringe communities of plains and of the montane to alpine levels; 6440 Alluvial meadows of river valleys of the Cnidion dubii; 9160 Sub-Atlantic and Central-European oak or oakhornbeam forests of the Carpinion betuli; 91E0 Alluvial forests with Alnus glutinosa and Fraxinus excelsior (Alno-Padion, Alnion incanae, Salicion albae) (priority habitat); 91F0 Riparian mixed forests of Quercus robur, Ulmus laevis and Ulmus minor, Fraxinus excelsior or Fraxinus angustifolia, along the great rivers (Ulmenion minoris). Life cycle, phenology and growth Seedlings of L. aestivum grow c. 1 cm in height in the first month and c. 2.5 cm in the second month. Seedlings and young plants can be easily recognized as they have only one or two leaves of 2–5 mm width until the second year. Plants stay in the subadult age class from 2 to 5/6 years, before they become adult flowering plants. Little is known about the longevity of the species, but Zagorska et al. (1997) recorded bulb aggregates older than 15 years, and plants have survived for more than 50 years in the botanical garden of the University of Halle, Germany (E. Jäger, pers. comm.). The bulbs may produce lateral daughter bulbs which remain attached to the mother bulb (Fig. 3), forming an aggregate of ramets. Each new ramet can reach the flowering stage. Up to four shoots can develop from a single bulb, growing inside the sheath of the mother plant (Fig. 4). In the plain of the river Po the ontogenetic cycle of L. aestivum lasts from October to June (Rossi and Dominione, 2005). Seeds of L. aestivum germinate in autumn (as in Germany; Irmisch, 1860), when soil and air humidity increase after the dry summer period. The epicotyl continues to grow slowly between January Fig. 3. Vegetative propagation in L. aestivum: the short underground caulis can generate new lateral bulbs that remain attached to the mother bulb. and February (Leonardi and Rossi, 2001; Çiçek et al., 2007). Shoots develop underground and emerge only when conditions become milder (Davis, 1966); long sheathing leaves appear aboveground in autumn, but foliage leaves start to develop in February–March. Inflorescences develop from February to March and the flowers open from late February to May, depending mostly on latitude (Guinea Lopez and Ceballos Jimenez, 1974). Fruits mature from May to June. After fruiting the leaves die off and the plant stays dormant until autumn. The subspecies pulchellum flowers 3–4 weeks earlier than the subspecies aestivum (Salisbury, 1807–1808; Tutin et al., 1980), probably due to the warmer climate of the Mediterranean Basin, where this subspecies occurs. Rarely, it can flower in December (Bolòs and Vigo, 2001). Spatial distribution of plants within populations The spatial distribution of L. aestivum plants within populations is influenced both by the ecological features of the micro-sites and by clonal growth. Within a population microsites with L. aestivum subsp. aestivum present are characterized by a significantly higher water content (48.1% vs. 44.7%, F1,165 = 4.5, p < 0.05), a lower dry matter content of sand (17.6% vs. 22.3%, F1,165 = 7.6, p < 0.01), but a higher of silt (59.7% vs. 55.6%, F1,165 = 8.2, p < 0.01), and a higher soil nitrogen content (0.55% vs. 0.49%, F1,165 = 5.0, p < 0.01) than microsites without the plant. Moreover, as a result of vegetative propagation plants usually grow in clumps and the spatial distribution is highly aggregated. Groups consisting of 10–20 bulbs can be formed from one initial bulb within 12–15 years (Zagorska et al., 1997). The density of plants can be very high (>1000 per m2 ), but is usually much lower. In the Po Plain some populations in 2007 consisted of more than 30,000 flowering scapes, while others consisted of a single flowering individual. Very small populations are common towards the margins of the range of the species (e.g. Greece, Bareka et al., 2003; Switzerland, Hess et al., 1967; France, Frédéric Médail, pers. comm.). Reproduction L. aestivum is a polycarpic species, whose flowers are pollinated by insects. Potential pollinators are long-tongued nocturnal Lepidoptera, diurnal Lepidoptera (Vanessa urticae) and Hymenoptera, Fig. 4. Shoots developing from a single basal caulis that remain included in the sheath of the mother plant. 325 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 60 1.0 50 Chi2 = 2008.4 0.8 r = 0.74 P < 0.01 Seed set (%) Probability of flowering p < 0.001 0.6 0.4 40 30 0.2 20 0.0 0 20 40 60 80 100 1 120 2 20 50 100 200 Fig. 6. The influence of the mean density of flowering shoots per m2 on seed set in populations of L. aestivum in the valley of the river Po in N-Italy. influenced by soil water availability at the time of seed ripening. The fruits are large and their weight bends the scape until the fruits lie on the ground. Between flowering and fruit maturation the scapes increase their length by up to a third, reaching a length of 92.7 ± 2.1 cm (n = 37). This could be interpreted as a means of dispersal to place the large and heavy seeds away from the mother plant. River water during flooding events may be an important means of dispersal, since fruits float on water and can thus be carried over long distances (Phillips and Rix, 1991). Fruits mature at the end of spring when the main European rivers, as a consequence of snow melting in the mountains, increase their flow and occasionally flood. 160 140 Seed mass (mg) such as bees (e.g. Anthophora pilipes, spring flower bee; Knuth, 1898–1904), Xylocopa violacea, and bumblebees. The flowers were observed to function as shelters for several species of beetles and spiders. Pollen is produced from 7 am to 6 pm (c. 0.1 mg per flower and day) and anthers dehisce over a period of 1–8 days (Percival, 1955). L. aestivum is self-incompatible (Knuth, 1909 and own observ.) and there is no apomixis. In N-Italy, most plants remain vegetative. Of more than 8000 plants studied, 30% flowered. Most flowering plants produce only a single flowering scape. In N-Italy, 82% of plants produced one flowering scape, 17% two scapes and only 1% three scapes. Each flowering scape produced on average 4.52 (±0.02) flowers (range 1–11), while total flowers of a plant were on average 5.47 ± 0.05 flowers (range 1–22). The probability of flowering increased with plant size (Fig. 5). In N-Italy the flowering probability of plants with leaves of less than 43 cm was 10%, while of those with leaves longer than 69 cm more than 50% flowered. Seed formation was studied in 404 fruits belonging to different plants in 20 populations in N-Italy. The mean number of ovules per fruit was 11.0 ± 0.21 of which 33.8% ± 0.07 developed into ripe seeds. This low seed set was mainly due to the large proportion of ovules (57.5% ± 0.89%) that were not fertilised and did not develop at all, and to a lesser degree to seeds that were aborted (8.7% ± 0.6). The mean number of seeds per fruit was 6.33 (±0.14). Seed set (22–57%) and thus the number of seeds per fruit (3.5–12.1) varied strongly among populations. Seed set of L. aestivum was not influenced by population size (r = 0.27, p = 0.24), but increased strongly with the density of flowering plants (Fig. 6). This was due to the reduction in the proportion of unfertilised ovules with density (r = −0.70, p < 0.01), while the proportion of seeds aborted was not influenced by density (r = 0.01, p = 0.99). This indicates that in L. aestivum reproduction is pollen limited in low-density populations. A similar behaviour was found by Diadema et al. (2004) for the related L. fabrei, a species endemic to France. Mean seed mass of L. aestivum varied strongly among populations, from 76 mg to 150 mg. It was not influenced by population size (r = −0.30, p = 0.21) or density (r = −0.14, p = 0.55), but increased strongly with the water content of the soil (Fig. 7), suggesting that seed development is 10 2 Length of longest leaf (cm) Fig. 5. Probability of flowering as a function of plant size (length of the longest leaf) in L. aestivum. The line is the best fit of a logistic regression. 5 Flowering shoots per m r2 = 0.51 p < 0.01 120 100 80 60 10 20 30 40 50 60 70 80 90 Soil water content (%) Fig. 7. The relationship between mean seed mass and water content of the soil in populations of L. aestivum in N-Italy. The line denotes a quadratic regression. 326 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 Herbivores and pathogens Fig. 8. Time course of germination of seeds of L. aestivum. The seeds were set to germinate at 20 ◦ C for 150 days and then exposed to a typical course of temperatures observed in the habitats of L. aestivum in N-Italy. Seed germination Collections of seeds were made at the time of natural dispersal (late May) from 14 natural populations in the Po Plain (N-Italy). Few days after collection, three replicates of 30 seeds for each population were sown on plain agar in 9 cm Petri dishes in the laboratory. Treatments were incubated in temperature and light controlled incubators using a 12 h daily photoperiod (4000 K, 20 W). Seed germination of L. aestivum depended strongly on temperature. After 100 days at 10 ◦ C and 15 ◦ C, no seeds had germinated. After the temperature had been increased to 20 ◦ C, seeds started to germinate c. 135 days after the start of the experiment, and after 260 days 75% of the seeds had germinated. At 20 ◦ C, germination started after c. 40 days and reached 79% after 150 days (Fig. 8). Germination varied strongly among populations (52–93%). Ungerminated seeds were then exposed to a cycle of temperatures to simulate the annual temperature cycle in the Po plain (Ottone and Rossetti, 1980). However, germination increased only at the end of this sequence, when seeds were transferred from 25 ◦ C to 20 ◦ C, reaching 100% in some populations. Similar results were obtained by Çiçek et al. (2007) who obtained 73% germination after 42 days at 22 ◦ C followed by 28 days at 20 ◦ C, but higher germination in a field experiment, where temperatures were higher. Stratification of the seeds for 3–7 weeks at 4 ◦ C induced dormancy (Çiçek et al., 2007). This behaviour suggests that seeds of L. aestivum only germinate at higher temperatures (>15 ◦ C), which are common after seed ripening in its area of distribution. However, summer drought could represent a limiting factor for the germination of Leucojum in the drier parts of its distribution. In Italy seedlings start to appear only in September–October, although seeds ripen and are shed already in June (Leonardi and Rossi, 2001). Response to competition L. aestivum subsp. aestivum can survive at a wide range of light levels from heavy shade to full sunlight. Even quite low levels of available light do apparently not reduce the vigour of the plants much (Kutbay and Kilinç, 1993). The early growth and flowering period avoid competition with most of the other plants of its habitats, mostly tall herbs such as Phragmites australis, Carex sp. pl., and shrubs like Rubus caesius. L. aestivum naturally produces some neurotoxic substances (see also ‘Biochemical data’ section) as a means of defence against herbivores and pathogens (Wink, 2009). However, the plant is frequently attacked by slugs and snails that eat scapes and fruits, mainly at the beginning of June. This can strongly reduce the number of seeds produced. In some large Italian populations, up to 100% of the developing fruits were destroyed. Fruit predation is common also in other species of Leucojum (Diadema et al., 2004). In some Italian populations we also observed strong damage to the flowering scapes by nutrias (Myocastor coypus). According to the Royal Horticultural Society (1996), larvae of the Narcissus Fly (Merodon equestris Fabricius, Syrphidae) may damage L. aestivum by eating bulbs. Aecidium leucoji (Uredinales, Basidiomycota) is a pathogenic fungus specific to L. aestivum that attacks leaves and fruits (Saccardo, 1899). The fungus occurs on L. aestivum in its ecidial form, but the reproductive form (Puccinia schmidtiana Dietel) is not known on L. aestivum; it was reported by Saccardo (1899) only on the leaves of P. arundinacea, a wetland plant species that grows frequently together with L. aestivum. The fungus is known for Europe since 1878 (Erb. Critt. Ital. ser. II n. 99), and its presence was confirmed for Italy, Germany and Hungary (Linhart, 1882). In the valley of the river Po, the fungus was present in 11 of the 26 populations of L. aestivum subsp. aestivum sampled in 2007. The probability of the presence of A. leucoji was not influenced by the density (Chi2 = 0.08, p = 0.78) of a population of L. aestivum, but large populations were more likely to be infested than small ones (Chi2 = 2.78, p < 0.1). The fungus had, however, no detectable influence on the performance and the reproduction of L. aestivum (fruit set, seed set). It is not known whether A. leucoji occurs also on L. aestivum subsp. pulchellum. In a population of L. aestivum subsp. aestivum (Corteolona, in the Province of Pavia, N-Italy), another fungus, Fusarium oxysporum (Fr.) Schltdl. was found on the fruits of the plant, in close proximity to A. leucoji. F. oxysporum, which has a world-wide distribution, is known to provoke wilt and damping off of the plants, and it generally affects the plants after a primary fungal infection (Cummins, 1971), here probably by A. leucoji. Systemic bulb infection represents a great obstacle to the micropropagation of L. aestivum and it is important to sterilize bulbs collected from wild populations as soon as possible to control the disease (Zagorska et al., 1997). Bulblets in the central part of the main bulb are usually less infected (Zagorska et al., 1997). Septoria malisorica Bubák is another pathogenous fungus which can affect the leaves of L. aestivum (Saccardo, 1913). Mycorrhiza L. aestivum is usually associated with arbuscular mycorrhiza (Harley and Harley, 1987). Physiological data The dormant bulbs of L. aestivum are fairly hardy and will withstand soil temperatures down to at least −5 ◦ C (Matthews, 1994). Flower initiation occurs at any temperature from 10 to 30 ◦ C, but the optimum temperature for flower initiation and further differentiation of floral organs is 20–25 ◦ C. In L. aestivum, further development of flower buds after the carpel formation stage is inhibited when plants are placed at or above 20 ◦ C (Mori et al., 1991b). Anthesis is stimulated by temperatures between 10 ◦ C and 15 ◦ C (Mori et al., 1991b), and can occur at 100% of relative humid- G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 ity, but it is limited by temperatures lower than 7 ◦ C and rainy weather (Percival, 1955). Biochemical data According to the International Plant Genetic Resources Institute L. aestivum is considered a Medicinal Plant (Baričevič et al., 2004), containing several pharmaceutically important alkaloids in its bulbs and leaves. These alkaloids are approved drugs for the treatment of a limited number of patients suffering from senile dementia of the Alzheimer type, poliomyelitis, alcohol and nicotine dependence (Moorman, 1999) and other neurological diseases (Thomsen et al., 1991; Schwikkard and van Heerden, 2002; Heinrich and Lee Teoh, 2004; Marco and do Carmo Carreiras, 2006). So far, about 20 alkaloids have been isolated from L. aestivum subsp. aestivum (Stefanov, 1990), belonging to five groups: N-allylnorgalanthamine, galanthamine, epinorgalanthamine, narwedine, and lycorine (Berkov et al., 2008). The most important of these are alkaloids of the galanthamine type, isoquinoline alkaloids with an inhibition effect on acetylcholinesterase (Diop et al., 2007). This inhibition is long-acting, selective, reversible, and competitive and produces beneficial effects even after the drug treatment has been terminated (Maelicke et al., 2001). Galanthamines for medical applications can be produced synthetically (Küenburg et al., 1999), but the most important source is bulbs of the Amaryllidaceae, such as Narcissus sp. pl. and Leucojum (Eichhorn et al., 1998). For this reason, bulbs of L. aestivum are still collected on a large scale in natural populations, causing the decline of the species (Çiçek et al., 2007). The alkaloid content of plants and the relative proportions of the main alkaloids (galanthamine and lycorine) vary among populations (Georgieva et al., 2007; Bogdanova et al., 2009) and depend on plant genotype (Stanilova et al., 2009). However, it has been demonstrated that L. aestivum has a high regenerative potential that could be used for micro-propagation. This kind of technique is increasingly applied to Leucojum (Girmen and Zimmer, 1987; Pavlov et al., 2007; Georgiev et al., 2009). Tissue and root hair culture has successfully yielded compounds identical to the alkaloids from natural sources (Diop et al., 2007). Stanilova et al. (1994) obtained about 630 plants from a single original plant of L. aestivum. Leaves are more suitable than bulbs for producing plants by micro-propagation, because it is not necessary to destroy the plants and because leaves are less contaminated by pathogens than bulbs. The cultivation experiments of Ayan et al. (2004) highlighted the potential of some plant growth regulators (PGRs) like GA3 and NAA, for obtaining larger plants. Plants grown under shaded conditions produce more alkaloids than those grown in full light (Kutbay and Kilinç, 1993; Ayan et al., 2004). Combining shading and PGR application could increase the production of galanthamines in in vitro cultures (Ayan et al., 2004). Galanthamine biosynthesis is also influenced by the concentration of nutrients in the medium. Georgiev et al. (2009) proposed as an optimal medium for the galanthamine production by a L. aestivum shoot culture the following concentration of nutrients: 4.50 g/L KNO3 , 0.89 g/L NH4 NO3 , 1.25 g/L (NH4 )(2) SO4 , 0.10 g/L KH2 PO4 and 60 g/L sucrose. Addition of the precursors tyrosine and phenylalanine in the medium enhanced the biosynthetic activity (Stanilova et al., 2009). Increasing N in the substrate resulted in higher bulb yield (Çirak et al., 2004). Human impact and use The main threat to L. aestivum is the collection of plants to extract galanthamines and other alkaloids used in the pharmaceu- 327 tical industry (Berkov et al., 2008). To extract 1 kg of galanthamine c. 1 tonne of bulbs are needed, as galanthamine concentrations are 0.1–0.2%. Large-scale collection has resulted in the decline of L. aestivum and further collection could lead to the disappearance of the plant from its natural habitats (Uherčíková, 1998; Ayan et al., 2004; Çiçek et al., 2007; Berkov et al., 2008). Turkey exports up to 6 million bulbs every year collected from natural populations for the production of galanthamines (Ayan et al., 2004). However, methods for the in vitro cultivation of the plant have recently been developed and may reduce the pressure on natural populations. Another threat to L. aestivum is the destruction of habitats. Due to the intensification of agriculture, many wetlands have been drained in the last decades and the habitats of L. aestivum have been destroyed. L. aestivum is widely cultivated as an ornamental plant in gardens and flowerbeds of temperate regions of the world, also outside of its natural range (e.g. in the USA), since L. aestivum is an attractive plant that can be easily transplanted and cultivated. A widely cultivated horticultural variety is “Gravetye Giant”, which grows more vigorously and has larger flowers than the wild type. Conservation status L. aestivum is not a very rare plant, but in recent decades it has declined due to the destruction of wetlands and harvesting for alkaloid production (Geneletti, 2007; Berkov et al., 2008). L. aestivum is included in several national red data lists. According to the IUCN Categories and Criteria (IUCN, 2001) it is listed as “Vulnerable” in the Carpathian area (Witkowski et al., 2003), Romania (Mardari et al., 2009) and Switzerland (Moser et al., 2002), and as “Least Concern” in Italy and Britain (Conti et al., 1997; Cheffings and Farrell, 2005). It is also included in the Russian Red Data Book (Iliashenko and Iliashenko, 2000), in the Red Data Book of Ukraine (ShelyagSosonko et al., 1996) and in the list of threatened species of the Pyrenees (Largier, 2003) that, however, do not use the IUCN criteria. Globally, it is probably not threatened yet. L. aestivum is legally protected in several countries and regions, such as Italy (Lombardy and Emilia-Romagna), Slovakia (Slovak Commission for Education and Culture, 1958) and France (Crellin, 2005), in Ramsar sites of Hungary (Central Agricultural Office, 2008), in Switzerland, Germany and Bulgaria. Here, since 1998 the harvesting of the plant is allowed only to obtain genetic material (Gussev et al., 2003). L. aestivum is not specifically mentioned in the most important international conventions (i.e. Berne, CITES, “Habitats” Directive), but some habitats where the species occurs are listed in the Ramsar Convention and in the “Habitats” Directive (see plant communities). Recently, as part of practical conservation efforts, L. aestivum has been used to restore wetland habitats (Rossi and Dominione, 2005). Plants propagated ex situ in the botanical garden of the University of Pavia have been used successfully to create new populations and reinforce existing ones in N-Italy (Leonardi and Rossi, 2001; Rossi and Dominione, 2005). Since native plants are not commonly marketed, in 2010 the Lombardy Region has funded the project “POT PLANT”, with the aim of creating a production chain of wild plants (including L. aestivum) to be used for gardening or translocation activities. In Bulgaria, to ensure a sustainable use of natural populations, ex situ conservation efforts were developed to assess the possibility of using seventeen natural populations as donors of germplasm, and to improve in vitro techniques and field cultivation practices like rapid bulb multiplication, and breeding of high reproductive clones (Gussev et al., 2003). For some pilot areas of southeastern France like the Port-Cros National Park, a dynamic management has been proposed to conserve L. aestivum subsp. pulchellum (Médail et al., 1995). 328 G. Parolo et al. / Perspectives in Plant Ecology, Evolution and Systematics 13 (2011) 319–330 Acknowledgements The authors are grateful to Dr. A. Mondoni (LSB – University of Pavia) for useful suggestions regarding the seed germination tests and for critical comments on the manuscript, to Dr. Erik Welk who kindly provided the distribution map of Fig. 2 (AG Chorology, Institute for Biology/Geobotany Halle, Germany), and to two anonymous reviewers for their valuable comments to the manuscript and their constructive suggestions. We also thank C. Amosso (Biella), J. Belotti (University of Pavia), P. Cauzzi (University of Pavia), S. Ciappetta (Domodossola), G. Decanis (Imperia), V. Dominione (Pavia), Dr. R. Gentili (University of Milan-Bicocca), A. Morini (Pavia), A. Podrini (University of Rome, La Sapienza), E. Vegini (University of Pavia) for their aid during field and laboratory work. We thank also Pepi Bareka (University of Patras), Patrick Grillas (Tour du Valait), Frédéric Médail (University of Marseille), Fred Rumsey (Natural History Museum, London), Arne Strid (University of Copenhagen), Gianluigi Bacchetta (University of Cagliari), Riccardo Guarino (University of Palermo), Harald Pauli and Michael Gottfried (University of Vienna) for information on the distribution of L. aestivum. The authors are grateful to Odile Weber and Paul Wilkin (R.B.G. Kew) and Fred Rumsey (Natural History Museum, London) for information about the type species. We thank Dr. Bruno Foggi (University of Firenze) for his help in the taxonomical interpretation of the subsp. pulchellum. We thank N. Ardenghi (University of Pavia) for the original drawings on Leucojum, and A. Picco and M. Rodolfi (University of Pavia) for the determination of pathogenic fungi. Last but not least, we thank F. Bonali (Cremona), G. Brusa (Varese), F. Giordana (Cremona), A. Petraglia (University of Parma), F. 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