ARTICLE IN PRESS Flora 202 (2007) 570–580 www.elsevier.de/flora Achene morphology and slime structure in some taxa of Artemisia L. and Neopallasia L. (Asteraceae) Agnieszka Kreitschitza,, Joan Vallèsb a Division of Plant Morphology and Development, Institute of Plant Biology, University of Wrocław, Kanonia 6/8, 50-328, Wrocław, Poland b Laboratori de Botànica, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Catalonia, Spain Received 18 August 2006; accepted 21 December 2006 Abstract We have examined slime cell distribution on the surface of the achenes of some Artemisia and Neopallasia taxa, as well as slime composition, envelope formation during the hydration, and slime relation to different morphological features and environmental factors. The results of the studies show a characteristic pattern of slime cells distribution, which could differ between taxa. The slime in the taxa studied belongs to the cellulose type and consists of two components i.e., pectins and cellulose. Although all fruits contain slime cells, not all of them show the slime envelope formation. Plants occurring in dry habitats (such as A. barrelieri) or annual species (such as A. annua) are characterised by a large amount of slime and a fast process of slime envelope formation. Slime production has not been observed in some polyploid populations (A. campestris and A. campestris ssp. sericea) and in two species occurring in relatively fertile habitats (A. verlotiorum, A. vulgaris). A reason for this may be either the immaturity of polyploid fruits leading to the production of a scarce, not detectable slime amount or, alternatively, the occurrence of not functional slime cells. Slime facilitates and stimulates the germination, as well as the adherence of the fruits to the ground or to animals (for dispersal). The slime could play important role in the distribution and colonisation of new habitats in many Artemisia taxa. r 2007 Elsevier GmbH. All rights reserved. Keywords: Achene; Slime cells; Cellulose slime; Pectins; Asteraceae Introduction Morphological and anatomical studies on the fruit and seed structure play an important role in systematics. Microstructural details of the seed and fruit coat make possible the distinguishing of taxa or the discovery of their affinities. This is especially useful in families in which identification of particular taxa is complicated, Corresponding author. Fax: +48 71 3754118. E-mail addresses: skowron@biol.uni.wroc.pl (A. Kreitschitz), joanvalles@ub.edu (J. Vallès). 0367-2530/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2006.12.003 e.g. in Portulacaceae, Scrophulariaceae, Polemoniaceae, Caryophyllaceae and Orobanchaceae (Johnson et al., 2004; Plaza et al., 2004; Zeng et al., 2004). Furthermore, observations of micromorphological features can also provide us with information about developmental strategies, adaptation to different environmental conditions and evolutionary tendencies within related groups of plants (Johnson et al., 2004; Plaza et al., 2004; Zeng et al., 2004). The presence of slime has been reported in diverse groups of organisms such as bacteria, algae, fungi, lichens and mosses (Mühlethaler, 1950). Slime is also ARTICLE IN PRESS A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 widespread in higher plants, especially in fruits and/or seeds in families such as Brassicaceae, Euphorbiaceae, Plantaginaceae, Linaceae, Malvaceae, and Lamiaceae (Baiges and Blanché, 1988; Huang et al., 2000; Mühlethaler, 1950; Western et al., 2000; Wojciechowska, 1961, 1966; Young and Evans, 1973). Within the Asteraceae family, slime has been reported, among others, in Filifolium (Mouradian, 1995), Achillea, Anthemis, Chrysanthemum (Grubert, 1974) and Artemisia (Boyko, 1985; Grubert, 1974; Huang and Gutterman, 1999a, b; Huang et al., 2000; Korobkov, 1973; Mouradian, 1995; Oganesova, 1981; Yakovleva et al., 2002). Pectins are the major component of slime, which is also present in all cell walls, mainly in the middle lamella. Pectins are mostly acidic polysaccharides with a special capacity for hydration. After wetting of dry mucilaginous seeds, the slime is released and forms a gel envelope (Fahn and Werker, 1972; Western et al., 2000). The classification distinguishes ‘‘true slime’’, consisting of pectins e.g. in Linum usitatissimum or Plantago psyllium, and cellulosic slime e.g. in Cydonia vulgaris and Salvia spp. (Frey-Wyssling, 1959; Mühlethaler, 1950). The latter type is characterised by the presence of an additional cellulosic skeleton, forming helicoidal thickenings, as in seeds of Cobea scandens (Fahn and Werker, 1972; Frey-Wyssling, 1959). Slime can be deposited between plasmalemma and the primary cell wall in the last stage of protoplast lifetime and then it dries out with fruit ripening. Slime formation can also result from secondary modifications occurring in the cell wall (Fahn and Werker, 1972). Different functions have been reported for slime. It plays an important role in the control of germination, mostly in plants that grow in the condition of water deficiency in arid and semiarid environments, thus facilitating imbibing and maintenance of the water. It can also delay germination due to impeded penetration of the oxygen. Slime helps in fruit or seed dispersal and in the defence against pathogens (Fahn and Werker, 1972; Huang and Gutterman, 1999a, b; Huang et al., 2000; Korobkov, 1973; Young and Evans, 1973; Young and Martens, 1991). Artemisia is one of the biggest and the most widely distributed genus of the Asteraceae family, occurring mostly in the Northern Hemisphere. Depending on the authors, it contains from 200 to ca. 500 species (Vallès and Garnatje, 2005 and references therein; Vallès and McArthur, 2001), including various life forms such as perennial shrubs and biennial or annual herbs. Most Artemisia taxa dominate in steppes, deserts or semideserts of Eurasia, North America and North Africa (Bremer, 1994; Gams, 1987; Polyakov, 1995; Vallès and McArthur, 2001; Żukowski, 1971). Many of them are common weeds; some are also used as food, 571 medicinal and ornamental plants. Neopallasia is an Asian monotypic genus segregated from Artemisia (Polyakov, 1995). The wide range of geographical distribution and variety of habitats in which Artemisia occurs may result in different adaptations to diverse environments. Adaptive features may be manifested by the changes in morphology and anatomy of different plant organs as well as in the cell structure and function, exemplified by the increase of ploidy level and nuclear DNA content (Garcia et al., 2004; Torrell and Vallès, 2001). Another important adaptive feature may be the slime presence in the fruits (Huang et al., 2000; Korobkov, 1973). Achenes of Artemisia are small, pappus lacking, ovoid to ellipsoidal in shape, thin-walled and usually glabrous but occasionally with some trichomes. Rows of myxogenic (mucilage-, slime-producing) cells can be present on the achene surface (Bremer, 1994). Some results on Artemisia achene sculpture have already been published (Korobkov, 1973; Ouyahya and Viano, 1984, 1990; Vallès and Seoane, 1992), but there is very scarce information on achene slime (Yakovleva et al., 2002) and almost no data on slime structure. The aims of this work have been (1) to examine the distribution of the slime cells on the achene surface, with possible differences between taxa, (2) to discern the slime structure, its composition and its formation after hydration and (3) to determine the relationship between slime presence and habitats, plant life form and ploidy level. Material and methods Plant material Achenes of 12 Artemisia and one Neopallasia taxa were used for the analysis of achene surface morphology and slime characteristics. Mature achenes were collected from natural plant populations. Plant vouchers are deposited in the herbaria of either A. Kreitschitz (Wrocław) or the Centre de Documentació de Biodiversitat Vegetal de la Universitat de Barcelona (BCN) or the Shrub Sciences Laboratory, Provo, UT (SSLP). The list of taxa, locations and collectors are presented in Table 1. Scanning electron microscopy (SEM) The sculpture of the achene surface was analysed on the SEM. Dry, mature achenes were mounted directly on the stubs using double-sided adhesive tape, coated with gold particles, and then observed in the SEM (JSM 5800LV JEOL and LEO435VP). ARTICLE IN PRESS 572 Table 1. A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 Location of the plants studied Taxon Genus Artemisia Subgenus Artemisia A. annua L. A. biennis Willd. A. verlotiorum Lamotte A. vulgaris L. Subgenus Absinthium A. absinthium L. Subgenus Dracunculus A. dracunculus L. A. campestris L. (2  ) Location Collector, date and herbarium Wrocław, Rzeszów (Poland) Rzeszów (Poland) Fogars de Tordera (Catalonia, Spain) Wrocław, Chrza˛ stawa Mała, Bolesławiec, Sobieszów (Poland) Kreitschitz (2003), AK Oklejewicz (2004), AK Oklejewicz (2004), AK Vallès (1998), BCN Kreitschitz (2004), AK Czeszów (Poland) Kreitschitz (2004), AK Wrocław (Poland) Oriola, Aranjuez (Spain) Żagań Brzezia Ła˛ ka, Ludgierzowice, Milicz, Miłochowice (Poland) A. campestris L. ssp. sericea (Fr.) Lemke and Rothm. Grzybowo, Ustka, Darłówko (Poland) Kreitschitz (2004), AK Torrell and Vallès (1996), BCN Gómez and Vallès (1994), BCN Da˛ browska (2002), AK Kreitschitz (2004), AK Kreitschitz (2004), AK Kreitschitz (2004), AK Klimczyńska (2002), AK Kreitschitz (2004), AK Subgenus Seriphidium A. barrelieri Besser Oriola (Spain) Vallès (1994), BCN Subgenus Tridentatae A. nova Nelson A. pygmaea A. Gray Utah (USA) Utah ( USA) McArthur (2004), SSLP McArthur (2004), SSLP Genus Neopallasia N. pectinata (Pallas) Poljakov Ulaan Baatar (Mongolia) Vallès (2004), BCN A. campestris L. (4  ) Slime identification by chemical reactions Microchemical reactions for cell wall components were carried out to identify the slime type. The following reagents were used: 0.1% aqueous solutions of methylene blue, ruthenium red, iodine in potassium iodide with sulphuric acid (I in KI+H2SO4), zinc chloroiodide (ZnClI) and alcohol solution of safranin (Braune et al., 1975; Broda, 1971; Filutowicz and Kużdowicz, 1951; Gerlach, 1972; Western et al., 2000). The images were taken using a light microscope OLYMPUS BX-50 connected with a SONY 3CCD colour video camera and graphic station Indy (SGI). Results Sculpture of achene surface The achene surface in all studied taxa but A. campestris ssp. sericea is glabrous and glossy. In A. campestris ssp. sericea long single trichomes occur on one end of the achene. They are fragile, easy to detach. The most external part of the fruit coat (achene coat) consists of two different types of cells, i.e. proper epidermal cells (Figs. 1–3B) and slime cells (Figs. 1–3A, B, C, Fig. 4K). Due to their arrangement, the achene surface is roughly sulcate. The proper epidermal cells formed ribs parallel to the long axis of the achene. These ribs can be evident as relatively wide strands (Figs. 1D, E, 2M), individual ribs (Figs. 1B, 2I) or separated cells (Fig. 2J). Sometimes the contours of the ribs can be very faintly marked, giving a smooth surface e.g. in A. absinthium (Fig. 1A). Much smaller than proper epidermal cells are the slime cells, here usually rectangular (Fig. 2M). They could be more irregular, oval to ovate or triangular, as in A. nova and A. pygmaea (Figs. 2K, 3B, C, 4K). Slime cells form ladder-like columns that are elongated in a parallel plane to the long axis of the achene. Ladder-like groups of slime cells can either evenly cover almost the entire surface of the achene, as in A. barrelieri and Neopallasia pectinata (Figs. 1C and 2N), or alternate with strands of the proper epidermal cells e.g. in A. absinthium, A. biennis and A. vulgaris (Figs. 1A, D and 2M). The surface of slime cells can be smooth ARTICLE IN PRESS A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 573 Fig. 1. (A–H) Achene surface showing the distribution of proper epidermal cells and slime cells; (A) Artemisia absinthium, (B) A. annua, (C) A. barrelieri, (D) A. biennis, (E) A. campestris (diploid: 2n ¼ 2x ¼ 18), (F) A. campestris (tetraploid: 2n ¼ 4x ¼ 36, achene from Brzezia Ła˛ ka locality), (G) A. campestris (tetraploid: 2n ¼ 4x ¼ 36, achene from Ludgierzowice locality) and (H) A. campestris ssp. sericea (tetraploid: 2n ¼ 4x ¼ 36, locality Grzybowo). White arrows indicate the proper epidermal cells, which occur as a smooth strips (A), ribs (D) or narrow individual cells (E). Black arrows show the slime cells, which are arranged in ‘‘ladder-like’’ strips: A, D, E, F and G. On the surface of the slime cells are visible delicate ripples (A–C, E). White arrows show the proper epidermal cells, black arrows – the slime cells. : cells are between arrows, : arrow shows the cell. (e.g. in N. pectinata – Fig. 2 N) or with delicate ripples as in A. absinthium or A. dracunculus (Figs. 1A, 2I). A distinct pattern occurs in A. nova achenes, where slime cells form small groups (Figs. 2J, 3B, 4K) scattered over the achene surface between proper epidermal cells. Both types of cells are evenly spaced along the long axis of the achene. The arrangement of the cells is rather the same in all studied taxa except A. nova, where the slime cells form short arrays (groups instead of long columns) ARTICLE IN PRESS 574 A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 Fig. 2. (I–P) Achene surface showing the distribution of proper epidermal cells and slime cells; (I) A. dracunculus, (J) A. nova, (K) A. pygmaea – the slime cells of irregular shape, (L) A. verlotiorum, (M) A. vulgaris, (N) N. pectinata; (O, P) cross section through the achene-the slime cells with a layer of slime-(O) A. absinthium and (P) A. campestris (tetraploid, locality Żagań), arrows show the thick layer of slime; white arrows show the proper epidermal cells, black arrows – the slime cells; : cells are between arrows, : arrow shows the cell. distributed among proper epidermal cells. In all the studied taxa, the differences concern particularly the form of the surface (smooth, with ripples or with folds, size of columns and cells), suggesting possible specific patterns for the different taxa. Further studies with a larger sampling might confirm whether these features could be useful for taxonomic purposes. Slime characteristics In the slime cells, a thick layer of slime is present (Fig. 2O, P). The Artemisia slime was determined to belong to the cellulose type. This type of slime represents a heterogenous system made of a pectinous matrix and a cellulose skeleton (Table 2). The presence of these ARTICLE IN PRESS A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 575 Fig. 3. (A–I) The slime cells, staining results and slime formation. (A) A. verlotiorum, (B) A. nova and (C) A. pygmaea-the slime cells of irregular shape, (D) A. annua pink, homogenously stained pectins are building a slime envelope, (E) N. pectinata – the beginning of staining, (F) N. pectinata very long cellulose threads (black arrows), (G) A. barrelieri, (H) A. campestris (tetraploid: 2n ¼ 4x ¼ 36, locality Brzezia Ła˛ ka) the coiled cellulose thread, (I) A. annua cellulose strands (black arrow). (A–C) slime cells without staining, after hydration, green arrows indicate the slime cells, (D–G) staining with ruthenium red (pectins detection), (H–I) staining with safranine (pectins detection). components was detected based on microchemical reactions. Treatments with different dyes revealed a characteristic staining pattern, this being in agreement with results known from literature (Table 2). Ruthenium red revealed a pink envelope around the achene (Fig. 3D–G). A red or orange–red coloration of slime was obtained with safranine (Figs. 3H, I, 4K). Methylene blue gave a very faint blue or almost undetectable colour; this metachromatic dye is also often used for cellulose identification, therefore results can be ambiguous. Cellulose was identified with four microchemical reactions (Table 2). Staining with ZnClI resulted in a delicate violet coloration of cellulose strands. In I in IK (Fig. 4L) and methylene blue (Fig. 4M–O) the cellulose strands were stained in blue. Staining reactions with ZnClI and I in IK are specific for cellulose detection, and also characteristic of cellulosic slime identification (Mühlethaler, 1950). A red (red–orange) colour of cellulose strands was obtained with safranine (Figs. 3H, I, 4J). Although the same colour was observed in the case of pectin staining, here the structural aspects of cellulosic strands were clearly visible, whereas ARTICLE IN PRESS 576 A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 Fig. 4. (J–R) Staining results and slime formation. (J) A. campestris (diploid: 2n ¼ 2x ¼ 18), (K) A. nova irregular shape of the slime cells, (L) A. campestris ssp. sericea (tetraploid: 2n ¼ 4x ¼ 36, locality Grzybowo), (M) A. annua, (N) A. biennis, (O) A. campestris (diploid 2n ¼ 2x ¼ 18), (P–R) N. pectinata, the beginning of the slime cell hydration (green arrows), (R) uncoiling of the cellulose threads (black arrows). (J, K) staining with safranine (cellulose detection), (L) I in IK+H2SO4 cellulose detection, (M–O) staining with methylene blue (cellulose detection). Black arrows indicate cellulose threads. pectin colour was spread homogenously within the envelope. Cellulose forms relatively thick helicoidal strands that after hydration can partially or entirely uncoil. After unwinding, these strands assume the shape of long, straight threads (Fig. 4M–O) forming a characteristic radial skeleton around the achene. Swelling and slime envelope formation Proper epidermal and slime cells form a transparent, delicate layer about 1–6 mm thick (Fig. 2O, P). This film can adhere tightly to the achene (e.g. A. vulgaris, A. verlotiorum) or enclose it loosely, forming a gap between the achene surface and the envelope. In the latter case, it makes for easy removal of the envelope, as in A. nova and A. pygmaea. The first step of the slime envelope formation is the swelling of pectin component of the slime (Fig. 4P). Then, the cell wall is perforated as a result of the increased pressure and the mucilaginous content is discharged (Fig. 4Q, R). Cellulose strands can remain coiled (A. absinthium, A. dracunculus) or, conversely, ARTICLE IN PRESS A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 Table 2. 577 Slime staining Staining Target Obtained colour Literature data References Ruthenium red Pectin Pink Pink, carmin-red, red Safranin Pectin Methylene blue Cell wall Pectin Red, orangered Red Not detected Brown to orangeyellow Red Blue Western et al. (2000); Filutowicz and Kużdowicz (1951); Gerlach (1972); Broda (1971) Gerlach (1972) ZnClI Cellulose Cellulose Blue Violet Violet, blue Violet, blue, dark blue I in IK+H2SO4 Cellulose Dark blue Dark blue to dirty blue Blue uncoil into the shape of long threads (Fig. 4M–O). Cellulose threads are usually shorter than the width of the achene. Only in Neopallasia uncoiled threads can be 3–4 times longer than the achene width, which is very characteristic of this genus (Fig. 3F). The process of swelling can be rapid, triggered immediately after wetting of the achene, e.g. in A. annua, A. barrelieri and N. pectinata. In some taxa, envelope formation last several minutes up to a few hours, e.g. in A. dracunculus and A. nova. Variation in slime envelope formation Although the slime cells are present in all studied taxa, swelling was not observed at all in A. vulgaris and A. verlotiorum. Additionally, achenes of A. campestris and A. campestris ssp. sericea coming from different localities and from different specimens show differences in the process of swelling and slime formation. Fruits of diploid (2n ¼ 2x ¼ 182Torrell et al., 2001) A. campestris, as well as of tetraploid (2n ¼ 4x ¼ 36 – Kreitschitz and Vallès, 2003) individuals from two localities (Żagań and Brzezia Ła˛ ka, Table 1), form a typical slime envelope (Fig. 3H), similar to other taxa. Conversely, achenes of tetraploid plants from four other localities (but including also some specimens from Brzezia Ła˛ ka) do not swell, although they clearly possess slime cells (Fig. 1G). If swelling takes place, it only affects few cells without visible slime envelope formation. A similar result was obtained for the tetraploid A. campestris ssp. sericea (2n ¼ 4x ¼ 36 – Kreitschitz and Vallès, 2003), where fruits from two localities (Grzybowo and Ustka) swelled (Fig. 4L), while those from another place (Darłówko) did not. Braune et al. (1975) Gerlach (1972); Filutowicz and Kużdowicz (1951) Broda (1971); Filutowicz and Kużdowicz (1951) Filutowicz and Kużdowicz (1951); Broda (1971); Braune et al. (1975); Gerlach (1972) Broda (1971); Johansen (1940); Filutowicz and Kużdowicz (1951); Braune et al. (1975); Gerlach (1972) Discussion As stated in the introduction, slime envelope formation is known in several plant families, including the Asteraceae. Concerning Artemisia it has been reported to date in A. arctica, A. subarctica, A. lagophus, A. lagocephala, A. scoparia, A. armeniaca, A. palustris, A. taurica, A. monosperma, A. absinthium and A. annua (Boyko, 1985; Grubert, 1974; Huang and Gutterman, 1999a, b; Huang et al., 2000; Korobkov, 1973; Mouradian, 1995; Oganesova, 1981; Yakovleva et al., 2002). Our results confirm the presence of slime in the two latter species and add six more taxa to the list of myxogenic Artemisia (A. barrelieri, A. biennis, A. campestris, A. campestris subsp. sericea, ;A. nova and A. pygmaea), these covering all the five large subgenera in which the genus is traditionally divided (Vallès and Garnatje, 2005 and references therein). In addition, this is the first record of the presence of slime in the genus Neopallasia. Previously, Young and Mayeux (1996) studying the seed ecology of some Artemisia species in subgenus Tridentatae did not report any species with slime cells in the achenes, although they have referred to mucilaginous trichomes in the seedlings. Our results reveal, however, the presence of slime cells, and show slime envelope formation in two members of this North American endemic subgenus (A. nova and A. pygmaea), in agreement with unpublished results on other taxa from this group (S. Garcia, personal communication). We also give the first report of the composition of the Artemisia achene slime in a large set of taxa; earlier only Yakovleva et al. (2002) mentioned the presence of cellulose as a component of slime in some Artemisia taxa. The structure of the slime cellulose threads is similar in all the species studied, which is logical taking into account their systematic closeness. The feature that differs between taxa is the length of these threads. ARTICLE IN PRESS 578 A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 They are much longer in Neopallasia (3–4 times the achene width) than in Artemisia (as long as the width of the achene). The monotypic genus Neopallasia was originally included in Artemisia and its taxonomic position is still disputable. Differences in slime envelope morphology may confirm the separate position of this genus. The ability to produce mucilaginous fruits, which is typical of many Artemisia taxa growing in dry habitats, was confirmed in our studies and is in agreement with data by Korobkov (1973) and Huang et al. (2000). Neopallasia grows in particularly dry habitats, where the presence of slime in the achene may be an advantageous adaptive feature facilitating germination. Additionally, the increase of the nuclear DNA content is thought to be an important adaptation to extreme environmental conditions in Artemisia. This is usually the result of polyploidization, but may also occur in diploids such as A. pygmaea (Garcia et al., 2004). The achenes of the taxa studied may differ in their slime content. We hypothezise that such differences may be associated with such factors as ploidy level, environmental conditions and life form. The achenes of some polyploid taxa, even those possessing slime cells, may be devoid of slime formation ability. This is the case of some tetraploid specimens of A. campestris and A. campestris ssp. sericea. In tetraploid plants of A. campestris, the slime cells are normally present on the achene surface but probably some fruits do not mature, or produce only a scarce amount of slime that is insufficient to form the envelope. Delayed maturation, absence of fruits and developmental changes are frequently the consequence of increased ploidy level due to the change in the length of the cell cycle. Polyploids often flower later than the diploids or sometimes do not form flowers at all (Bayer, 1998; Mizianty, 1994; Solbrig, 1977). For instance, tetraploid plants of A. abrotanum rarely flower and are usually not able to develop fruits (Kreitschitz, 2003). It is worth noting that Mouradian (1995) included A. campestris into the group devoid of slime cells. With the present data, we confirm not only the presence of slime cells in this species, but also the slime envelope formation in several populations as well. Similarly, only a few tetraploid specimens of A. campestris ssp. sericea ð2n ¼ 4x ¼ 36Þ (Kreitschitz and Vallès, 2003) were able to swell and to form a distinct slime envelope. The number and distribution of slime strands on the achene surface as well as their morphology may provide us with additional information about the differences in the slime amount in taxa studied. The slime layer deposited in the slime cells makes them higher than the proper epidermal cells. Pectin dessication may cause shrinkage of the cell walls and, as a result, cell obliteration, evident as ripples and folds on the achene surface. Based on the fold structure in slime cell strands in A. annua and A. barrelieri, it could be expected that these species possess an enormous amount of slime. On the other hand, delicate lines on the slime cell strands in A. absinthium, A. dracunculus and A. biennis suggest scarce slime production. Neopallasia pectinata is also potentially rich in slime, as its achenes are covered by numerous convex strands of slime cells. Consistently, microstaining reactions do reveal a wide envelope around the achenes of Neopallasia. Slime formation on the fruit and/or seed surface is known to be an ecological adaptation to limited availability of water (Huang and Gutterman, 1999a; Huang et al., 2000; Korobkov, 1973; Young and Martens, 1991). Differences in the slime amount can result from habitat diversity. Such a relationship was reported in the Lamiaceae (Mosquero et al., 2004). Apart from the case of tetraploid specimens of A. campestris discussed above, only in the cases of A. vulgaris and A. verlotiorum does the slime envelope remain unformed, although the slime cells are present on the achene surface in both species. These two taxa occur in diverse habitats, mostly growing in relatively fertile and wet environments such as river and lake banks, field margins, gardens and ruderal places (Cullen, 1975; Gams, 1987; Żukowski, 1971). It can be assumed that, in such environments, slime presence may not be necessary to assure seed germination. Therefore, the slime cells in these taxa may not mature or function properly. Similar data were given for Prunella (Lamiaceae), where the absence of slime or its low production may result from not completely formed or not functional slime cells (Mosquero et al., 2004). On the other hand, in Artemisia dracunculus, growing often on river banks (Gams, 1987; Rothmaler, 1987; Żukowski, 1971), the presence of a slime envelope may play an important role in the dispersal of this species. The slime can reduce the specific weight of diaspores, thus facilitating their transportation with the water current (hydrochory) (Fahn and Werker, 1972; Huang et al., 2000; Young and Evans, 1973). Taxa, such as A. absinthium, A. campestris, A. campestris ssp. sericea, A. pygmaea, A. barrelieri and N. pectinata, occurring in less fertile and often arid sandy locations (Cullen, 1975; Gams, 1987; Polyakov, 1995), are characterised by the presence of numerous strands of slime cells. The slime cells cover the entire achene surface and produce a distinct slime envelope in these taxa. The slime envelope facilitates the adherence of achenes to the soil surface, helps retain the water around the fruit and makes germination easier (Fahn and Werker, 1972; Huang et al., 2000; Young and Martens, 1991). As Baiges et al. (1991) stated for Euphorbia species, the slime in the dispersal units (seeds or fruits) allows the plants, which produce it, to create appropriate conditions for establishment and germination. ARTICLE IN PRESS A. Kreitschitz, J. Vallès / Flora 202 (2007) 570–580 In some particular cases, production of slime on the fruit and/or seed surface may also be an adaptation to ruderal, disturbed environments (Young and Evans, 1973). The presence of a slime envelope is associated with a short life cycle and facilitates a quick colonisation of such places. We confirmed this kind of adaptation in annual taxa like A. annua and A. biennis, growing in disturbed areas, and N. pectinata, occurring in semideserts (Cullen, 1975; Polyakov, 1995; Żukowski, 1971). Similar adaptive mechanisms are present in many common annual weeds colonising ruderal habitats, e.g. Lepidium flavum, L. nitidum, Plantago lanceolata, Cardaria draba (Young and Evans, 1973) and Arabidopsis thaliana (Western et al., 2000). Slime is an important factor regulating germination and development of seedlings. It was reported that removal of the slime envelope from seeds before germination may affect seedling development and cause the decrease of their size (Huang and Gutterman, 1999a, b; Huang et al., 2000). The presence of the slime is also advantageous as regards the protection of fruits and seeds from ingestion by animals and from pathogen infection. It facilitates dispersal (zoochory) and anchors seed to the soil surface (Huang et al., 2000). It can be hypothesised, that for the latter function a heterogenous type of slime, consisting of two components, i.e. pectins and cellulose, is more beneficial. Cellulose threads may strengthen the anchorage, thus keeping the achene in the soil. This is apparent in the extremely long threads of N. pectinata, which can protect the achene against wind dispersal. Acknowledgements The authors would like to thank: Prof. J. Da˛ browska (Wrocław University), Dr. E.D. McArthur (USDA, Provo, Utah), Dr. M. Klimczyńska-Szymura (Agricultural University Wrocław), Dr. K. Oklejewicz (Rzeszów University), M. Torrell and Dr. A. Gómez (Universitat de Barcelona) for their help in the collection of material, Msc. A. Kamińska (Technical University of Wrocław) and Msc. K. Heller (Agricultural University Wrocław) for the SEM pictures preparation, Dr. T. Garnatje (Institut Botànic de Barcelona), S. Garcia (Universitat de Barcelona) for comments which improved the manuscript, Dr. E. Gola and Dr. A. Banasiak (Wrocław University) for their critical revision of this work and S. Pyke (Jardı́ Botànic de Barcelona) for the correction of the English language. 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