Abnormalities in pistil development result in low seed set in Leymus chinensis (Poaceae)

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1 Flora 201 (2006) Abnormalities in pistil development result in low seed set in Leymus chinensis (Poaceae) Nianjun Teng a,b, Tong Chen a,b, Biao Jin c, Xiaoqin Wu a,b, Zehao Huang a, Xiaoguan Li a,b, Yuhua Wang a,b, Xijin Mu a, Jinxing Lin a, a Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing , China b Graduate School of the Chinese Academy of Sciences, Beijing , China c Department of Horticulture, Agricultural College, Yangzhou University, Yangzhou , China Received 8 October 2005; accepted 22 December 2005 Abstract Megasporogenesis and megagametogenesis in Leymus chinensis and the callose deposition during these developmental processes were investigated. In addition, morphological and histochemical studies of pistils at anthesis and pollen behavior on the stigmas after pollination were examined. The results indicate that embryo sac development and callose deposition pattern of this grass follow the archetypal Polygonum type. Nearly half of the pistils developed abnormally in megagametogenesis, while only 8.6% of abnormalities occurred in megasporogenesis. Over 47% of pistils at anthesis were abnormal in appearance. By 24 h after anthesis, many pollen grains had germinated on the stigmas of normal pistils. The high percentage of abnormal pistils and their low capacity to capture pollen grains may be the main factor in the low seed set of L. chinensis. r 2006 Elsevier GmbH. All rights reserved. Keywords: Callose deposition; Embryo sac development; Leymus chinensis; Low seed set; Pistil development Introduction Leymus chinensis (Trin.) Tzvel. (Poaceae) is a perennial rhizome grass that is widely distributed in the eastern end of the Eurasian steppe, from North Korea westward to Mongolia and northern China and north-westward to Siberia (Kuo, 1987). This grass is economically and ecologically important because it is rich in vitamins, high quality protein, minerals, and carbohydrates; moreover, it grows rapidly and is highly tolerant of arid conditions (Huang et al., 2002). Corresponding author. Fax: address: linjx@ibcas.ac.cn (J. Lin). However, its low rate of sexual reproduction drastically limits its propagation (Huang et al., 2002). The average rate of seed set in this species is about 56.7% in artificially constructed grasslands and less than 25% in natural grasslands (Wang, 1998). Because L. chinensis is an economically and ecologically important grass, it has received considerable attention in recent decades (Pan and Sun, 1986; Wang, 2001; Song et al., 2003). However, most studies of this species have focused on the effects of ecological factors on its seed production, such as climate (Wang et al., 2003), water use (Song et al., 2003), and nutrient uptake (Wang, 2001). Only a few investigations of the sexual reproduction of this species have been reported. Pan and /$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi: /j.flora

2 N. Teng et al. / Flora 201 (2006) Sun (1986) and Ma et al. (1984) found that over 83.5% of the pollen grains were viable and concluded that pollen grain viability is not a factor in the low seed yield. We have previously investigated the pollination, microsporogenesis, and callose deposition during microsporogenesis in this species, and have found that pistil receptivity and pollen longevity may have slight effects on the low seed set but are not the main factors. We have also observed that microsporogenesis and callose deposition generally appear normal, and have thus speculated that male reproductive development is not a factor in the low seed output (Huang et al., 2002, 2004; Teng et al., 2005). Therefore, the causes of the low rate of sexual reproduction in this grass are still unclear, largely because many aspects of the reproductive processes, especially the female reproductive development, remain unknown. So far, only a few investigations on the female reproductive processes have been reported, and almost no reports have examined the relationship between these processes and the low seed production in this vital species (Wei and Shen, 2003). As a continuation of our studies of the past several years (Huang et al., 2002, 2004; Teng et al., 2005; Yang et al., 2001), this study is part of a series examining sporogenesis and gametogenesis, callose deposition during these developmental processes, pollination and fertilization, and embryogenesis in L. chinensis. Our overall aim was to identify the main causes of the low seed yield and to provide knowledge that will be useful in increasing the seed production of this important grass. Materials and methods Plant material and growth conditions Plants of L. chinensis growing in the south of the Xilin River basin, Inner Mongolia, China ( N, E), at approximately 1265 m ASL (above sea level) were used in the study. The average rate of seed set is less than 30%. This site has a temperate, semiarid climate with a mean annual precipitation of 350 mm and a mean annual temperature of 0.3 1C (Chen, 1988). Examination of pistil development Spikes were collected daily from June 8 to 28, 2002 and immediately immersed in FAA solution (formalin 5 ml, acetic acid 5 ml, alcohol 70% 90 ml) until use. Pistils were dissected from florets, dehydrated through a graded series of ethanol solutions, and then embedded in paraffin wax. Sections were cut to a thickness of 8 10 mm and then stained with Heidenhain s hematoxylin or 1 mg/ml 4,6-diamidino-2-phenylindole (DAPI) in TAN buffer (Fujie et al., 1994) to examine pistil development or with decolorized aniline blue (0.01%, w/v; Worrall et al., 1992) to investigate callose deposition during the reproductive processes. The samples were observed under a fluorescence microscope with excitation filter BP , chromatic beam splitter FT 460 and barrier filter LP 470 for aniline blue staining, and with excitation filter BP 365, chromatic beam splitter FT 395 and barrier filter LP 420 for DAPI staining and with bright-field illumination for hematoxylin staining (Zeiss Axioskop 40). Digital images were captured using an Axiocam MRC camera. Examination of abnormal pistils at anthesis Spikes without dehiscing anthers just prior to anthesis were randomly selected from L. chinensis plants, and then fixed in FAA. Florets were collected from the upper, middle and lower positions of each spike, respectively, and then dissected under a dissecting microscope. The pistils dissected from florets were classified into normal and abnormal types. The abnormal pistils were sequentially divided into four classes according to their appearance: abnormal color (yellow or brown); abnormal stigmas that have not developed normally or have degraded; small sized pistils (less than half the size of normal pistils); and pistils with a flat ovary. Micrographs of normal and abnormal pistils were taken under a dissecting microscope with a digital camera. In addition, some pistils were examined using a scanning electron microscope according to the procedure described by Mundry and Stu tzel (2004). Furthermore, paraffin sections of abnormal pistils were also stained with decolorized aniline blue to investigate callose deposition. Pollen behaviors on pistils after anthesis Spikes at 24 h after anthesis (post-pollination) were fixed in ethanol: acetic acid (3:1) for approximately 30 min and then stored in 70% ethanol until use. The pistils dissected from the florets were softened overnight in 1 N NaOH and washed in distilled water three times. After that, the samples were stained with decolorized aniline blue, squashed and then examined and photographed using the above methods. Results Flower morphology L. chinensis flowers are hermaphroditic and arranged in compound spikes (Huang et al., 2004). The length

3 660 ARTICLE IN PRESS N. Teng et al. / Flora 201 (2006) of spikes ranges from 11.7 to 14.2 cm (average cm). Each spike comprises spikelets. There are approximately flowers or florets per spikelet. Each flower has an outer bract (lemma) and an inner bract (palea) at its base, and contains two lodicules and one pistil surrounded by three anthers (Fig. 1). The mature pistil has two feathery stigmas and a hairy ovary that contains one anatropous ovule (Fig. 2).

4 N. Teng et al. / Flora 201 (2006) Megasporogenesis and megagametogenesis Megasporogenesis and megagametophyte development generally took place from June 13 to 28 (Table 1). The ovule is anatropous, bitegmic, and tenuinucellar and contains one megaspore mother cell in the nucellus (Fig. 10). Megasporocytes were first observed in buds collected on June 13. The megasporocyte is easily distinguished from the surrounding cells because of its larger size, prominent nucleus, and distinct vacuole (Fig. 10). Next, the megasporocyte undergoes two successive divisions to form four haploid megaspores. The first division is transverse, resulting in two dyad cells with conspicuous nuclei (Fig. 11). The second division usually gives rise to a linear tetrad (Fig. 12), and occasionally a T-shape tetrad. Only the chalazal megaspore continues to develop into a functional megaspore, and the other megaspores gradually degenerate (Figs. 12 and 13). The functional megaspore then develops into a vacuolated embryo sac with a conspicuous nucleus in the center (Fig. 14). The young embryo sac develops into a binucleate, then tetranucleate, and finally octanucleate embryo sac (Figs ). During this developmental process, the size of the embryo sac increases greatly. In the mature embryo sac, the egg apparatus consists of an egg cell and two synergids at the micropylar end, a binucleate central cell, and usually three to six antipodal cells near to the chalazal end (Figs. 17 and 18). Table 1 shows a high percentage of abnormalities occurred during pistil development. However, less than 10% of pistils developed abnormally in megasporogenesis, whereas nearly 50% of abnormalities were observed in megagametogenesis. Those abnormalities were mainly characterized by the degeneration of embryo sac or the degradation of ovule tissues, similar to those observed in abnormal pistils at anthesis (Figs. 20 and 21). Dynamic changes in callose deposition Callose initially forms around the megaspore mother cell during the meiotic prophase. By the first meiotic Table 1. Developmental stages of megasporogenesis and megagametogenesis of Leymus chinensis and abnormalities of pistil development Date Stage of development June 13 20, 2002 Megasporogenesis 8.6 June 21 28, 2002 Megagametogenesis 48.5 Abnormalities of pistil development (%) metaphase, the entire megasporocyte is enveloped in a callose-containing wall (Fig. 22). At the dyad stage, the dyad is completely enclosed by thick walls that exhibit strong callose fluorescence, and the two dyad cells are separated by a thick cell plate that also shows strong fluorescence (Fig. 23). However, when the mother cell reaches the tetrad stage, the callose is unevenly deposited in the walls of the tetrad (Figs. 24 and 25). In general, the external transverse wall of the chalazal megaspore emits weak or no fluorescence (Fig. 24), whereas the other three megaspores are still enveloped by a callose-containing wall. In addition, the transverse walls usually exhibit stronger callose fluorescence than the side walls adjacent to the somatic cells of the surrounding tissue (Figs. 24 and 25). Next, the callose around the tetrad degrades in the direction from the chalaza to the micropyle (Fig. 26). By the time the chalazal megaspore has differentiated into the mother cell of the embryo sac, the other three megaspores have completely degenerated and very weak or no callose fluorescence is visible around the tetrad (Fig. 27). Thus, at this stage, the wall of the mononucleate embryo sac is devoid of callose, and only slight callose fluorescence is detectable in the surrounding somatic tissues. During the development of the mononucleate embryo sac into a binucleate (Fig. 28), then a tetranucleate (Fig. 29), and finally an octanucleate (Fig. 30) embryo sac, callose does not appear. Therefore, callose only forms during megasporogenesis and begins to degrade at the tetrad stage, and no callose is synthesized during megagametogenesis. Many abnormal patterns of callose deposition were observed during pistil development. However, most of Figs The morphology of flower and pistil of Leymus chinensis and pollen grains on the stigmas. 1. A complete flower, showing three anthers, one pistil with two feathery stigmas, an outer bract and an inner bract. 2. A normal pistil with two feathery stigmas and a hairy ovary. 3. Abnormal pistils in appearance at anthesis are often characteristic of small size, abnormal stigmas or flat ovaries and so on. 4. Normal pistils at anthesis are characterized by a full and hairy ovary, two feathery stigmas. 5. SEM micrograph of a normal ovary, and the ovary is full. 6. SEM micrograph of a abnormal ovary, and the ovary is flat. 7. Many pollen grains germinated on the stigma of normal pistil and pollen tubes grew down through the transmitting tissues of the stigma. Callose was formed during pollen tube elongation and visualized after staining with aniline blue. 8. Although a few pollen grains were sometimes captured on the abnormal stigmas, they did not germinate or they germinated abnormally. 9. A lot of pollen grains were deposited on the stigmas of a normal pistil. Abbreviations: An, anther; Ib, Inner bract; Ob, Outer bract; Ov, Ovary; Pg, Pollen grain; Pi, Pistil; Pt, Pollen tube; St, Stigma. Bars: Fig. 1: 1 mm; Fig. 2: 400 mm; Figs. 3 and 4: 800 mm; Figs. 5 7: 130 mm; Fig. 8:65mm; Fig. 9: 250 mm.

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6 N. Teng et al. / Flora 201 (2006) them took place during the late stages of pistil development, similar to those observed in abnormal pistils at anthesis (Figs ), and only a few abnormal patterns of callose deposition were observed in megasporogenesis, similar to the figures shown in Figs Abnormalities of pistils at anthesis In comparison with the morphological characteristics of normal pistils (Figs. 2, 4 and 5), the aberrant pistils were characterized by their small size, abnormal color, irregular stigma, or flat ovary (Figs. 3 and 6). Over 47% of the pistils of over 5000 flowers dissected at anthesis were aberrant. The percentage of abnormal florets in the middle position of a spike was about 36%, which was lower than the 58% and 55% abnormality rates of florets in the upper and lower parts, respectively (Fig. 37). It is evident that about 55% of the abnormal pistils belong to abnormal stigmas, about 20% of which are the small pistils in size, about 15% with flat vary and about 10% with abnormal colors (Fig. 38). Observations of hundreds of paraffin sections showed that aberrant pistils lacked an embryo sac, or the embryo sacs were devoid of contents or had degenerated abnormally (Figs ). Aniline blue staining also often revealed irregular callose deposition in the ovules of the abnormal pistils. For example, callose accumulated on the inner surfaces of the embryo sacs of aberrant pistils (Figs. 31 and 32) or in degenerating ovule tissues (Fig. 33). The layer of callose was generally thicker on the surfaces near the chalazal region than in other parts. Some aberrant pistils had no embryo sacs and showed irregular callose deposition in the nucellar tissues (Figs ). Pollen behavior on pistils after anthesis In general, a large number of pollen grains had deposited on the stigmas of normal pistils by 24 h after anthesis (Fig. 9). Other than this, there were no significant differences in appearance between pistils just prior to anthesis and those at 24 h after anthesis (Figs. 2 and 9). Aniline blue staining showed that many pollen grains germinated on the stigmas of normal pistils, and pollen tubes grew down through the transmitting tissues of the stigmas (Fig. 7). However, very few pollen grains were observed on the stigmas of aberrant pistils. Even though some pollen grains were sometimes deposited on the abnormal stigmas, they either did not germinate or germinated abnormally (Fig. 8). Discussion The embryo sac of L. chinensis follows the Polygonum type of development (Johri et al., 1992; Reiser and Fischer, 1993), as do those of all other Poaceae species that have been examined (Johri et al., 1992) and over 80% of angiosperms (Batygina, 2002). In most L. chinensis florets, microsporogenesis and megasporogenesis occur nearly simultaneously within the floret. In our study, microsporogenesis and pollen development took place from June 12 to 26 (Teng et al., 2005), and megasporogenesis and megagametogenesis generally occurred from June 13 to 28. When the functional chalazal megaspore still lacks a vacuole, the microspore has a single nucleus, and when the embryo sac has seven cells, the pollen grain has three cells. Callose, a specialized wall material that is composed of b (1 3)- linked glucose polymers, fluoresces brightly when stained with decolorized aniline blue (Bhatia and Malik, 1996; Lu et al., 2003). The pattern of callose deposition during pistil development in L. chinensis is similar to that reported in a previous study of plants that form the Polygonum type of embryo sac (Rodkiewicz, 1970). Ovule sterility is common in flowering plants and leads to low seed set in many seed plants (Wiens et al., 1987). Chaenomeles japonica plants with the highest Figs Megasporogenesis and megagametogenesis in Leymus chinensis and anatomical structures of abnormal pistils at anthesis. 10. At the stage of megasporocyte, the megasporocyte is characterized by larger size, prominent nucleus and distinct vacuole. 11. At the dyad stage, the two dyad cells are separated by a cell plate and easily discriminated from the surrounding somatic cells. 12. At the tetrad stage, the functional megaspore near the chalazal end persists and the megaspores adjacent to the micropylar end are under degeneration (white arrows). 13. The functional megaspore is characterized by a conspicuous nucleus in the center and dense cytoplasm, whereas the three non-functional megaspores have degenerated completely. 14. The mononucleate embryo sac possesses a prominent nucleus and a small vacuole. 15. The binucleate embryo expands greatly in size and a big vacuole appears. 16. The tetranucleate embryo sac possesses a conspicuous vacuole in the center. The two nuclei are situated near the chalazal end and the other two near the micropylar pole. 17. At the stage of octanucleate embryo sac, the egg apparatus consists of an egg cell and two synergids at the micropylar end, a binucleate central cell and usually three to six antipodal cells near the chalazal end. 18. There are more than three antipodal cells near the chalazal end (Staining with DAPI). 19. The ovary of abnormal pistil lacks an embryo sac. The white arrow shows that the megaspore mother cell had degraded and ceased to develop during the early developmental stage. 20. The embryo sac has degenerated. 21. The central cell still persists, but the egg apparatus has degenerated. Abbreviations: Ac, antipodal cell; C, chalaza; Cc, central cell; Ea, egg apparatus; Fm, functional megaspore; Ii, inner integument; M, megasporocyte; Oi, outer integument; V, vacuole. Bars: Figs : 25mm; Figs : 65mm.

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8 N. Teng et al. / Flora 201 (2006) Total number of florets Number of abnormal florets Percentage of abnormal florets 70 Number of florets Percentage of abnormal florets (%) Abnormalities of pistils (%) Upper Middle Lower Total Position of spike Fig. 37. Percentage of abnormal pistils in the different positions of spikes at anthesis. 0 0 Abnormal Color Abnormal stigmas Small pistils Classes of abnormal pistils Flat ovaries Fig. 38. Percentage of different classes of abnormal pistils at anthesis. percentage of abnormal pistils have the lowest fruit set (Kaufmane and Rumpunen, 2002). We found that most irregularities appeared in megagametogenesis; only a few abnormalities occurred in megasporogenesis. In addition, nearly 50% of pistils at anthesis were abnormal in appearance. Therefore, we speculate that the high percentage of abnormal pistils is the major cause of the low seed yield of this grass. The high rate of abnormal pistils may be caused by environmental factors, such as high temperature and arid climate. Callose deposition is usually correlated with ovule abortion (Arbeloa and Herrero, 1991; Rodrigo and Herrero, 1998). Faulty timing in the deposition or degradation of callose may influence sporogenesis and gametophyte development in many angiosperms, eventually resulting in gametophyte sterility (Rodrigo and Herrero, 1998; Rosellini et al., 1998; Vishnyakova, 1991; Worrall et al., 1992). In apricot, one of the two ovules in the ovary generally degenerates following irregular callose deposition around the ovule, and the other usually develops into a seed (Arbeloa and Herrero, 1991; Rodrigo and Herrero, 1998). In alfalfa, the first sign of sterility is callose deposition in the nucellus cell walls surrounding the sporogenous cells, whereas no trace of callose is present in ovules of fertile plants at the same stage (Rosellini et al., 2003). The irregular deposits of callose around the degenerating ovule before the appearance of morphological symptoms of ovule degeneration are thought to prevent the transport of metabolites from the surrounding somatic tissues into the ovule, thus arresting ovule development and eventually resulting in ovule degeneration, because the degenerating ovule lacks nourishment for its development (Mogensen, 1975; Pimienta and Polito, 1982). In our study, many irregularities in callose deposition were observed in the ovules of abnormal pistils, an Figs Callose deposition during megasporogenesis and megagametogenesis of Leymus chinensis and the abnormalities of callose deposition in the ovaries of abnormal pistils at anthesis. 22. Megaspore mother cell is enclosed in a thick layer of callose wall. 23. The dyad is enveloped by a callose wall and the two dyad cells are separated by a callose wall. 24. The linear tetrad is unevenly surrounded by callose wall. The black arrow shows that the chalazal megaspore is partly wrapped by a callose wall and the chalazal side wall is nearly free of callose, while the other three megaspores adjacent to the micropyle are still encompassed by a callose wall. 25. A T-shaped tetrad is surrounded by a callose wall. 26. At the late stage of tetrad, callose in the wall degraded in the direction from the chalaza to the micropyle. The white arrow demonstrates that the wall of chalazal megaspore has been devoid of callose and black arrow shows that the megaspores near to the micropyle are still partly surrounded by callose wall No callose appears during the various stages of embryo sac development. 31. The embryo sac is empty and black arrows indicate that callose mainly accumulated along the inner surfaces, especially in the chalazal and micropylar regions. 32. There are some contents in the embryo sac, but the inner surfaces, especially the chalazal region (black arrows), emits strong callose fluorescence. 33. The embryo sac has degenerated and callose fluorescence (black arrows) can be observed in the residues of embryo sac and its surrounding tissues Various irregularities of callose deposition take place in the ovules of abnormal pistils at various developmental stages of ovules. From these pictures, it can be speculated that some abnormal pistils at anthesis do not possess embryo sacs at all. Bars: Figs. 31 and 34 36: 20mm; Figs : 25mm; Figs. 32 and 33: 40 mm.

9 666 ARTICLE IN PRESS N. Teng et al. / Flora 201 (2006) observation that corroborates previous findings that ovule abortion is usually accompanied by abnormal callose deposition. Similarly, we previously found that a low rate of abnormalities in callose deposition during microsporogenesis in L. chinensis corresponds well with a high rate of viable pollen grains (Teng et al., 2005), which in turn demonstrates that callose deposition is also correlated with sporogenesis and gametogenesis. Therefore, we believe that the abnormal deposition of callose during female developmental processes is a predictor of ovule abortion in L. chinensis. Pollination is the primary step in seed set, and failures in pollination can occur at various steps during the release, transport, and deposition of pollen grains from the anther to the stigma. Such failures are thought to be widespread in plants and a common cause of low crop yields (Wilcock and Neiland, 2002). Too few or lowquality pollen grains delivered to a stigma may increase the risk of pollination failure and reduce the seed set (Aleemullah et al., 2000; Baker et al., 2000; Wilcock and Neiland, 2002). In addition, the structures of the stigma and its secretory products may influence the adhesion, hydration, and germination of pollen grains (Heslop- Harrison and Heslop-Harrison, 1983, 1985; Heslop- Harrison, 2000; Sanchez et al., 2004). The results presented here indicate that the feathery stigmas of normal L. chinensis pistils usually captured many pollen grains, which germinated by 24 h after anthesis. Numerous pollen tubes then penetrated into the feathery stigma tissues, growing into the stigma and the style. However, very few pollen grains appeared on the stigmas of aberrant pistils, and the pollen grains that were deposited on these stigmas usually either failed to germinate or germinated abnormally. We speculate that normal and abnormal pistils possess different abilities to capture pollen grains due to the following reasons. First, in comparison with aberrant pistils, normal pistils usually have stigmas with larger surface areas, which may facilitate the deposition of pollen grains. Second, the stigmas of normal pistils may secrete chemical products that facilitate the adhesion and germination of pollen grains, whereas the abnormal stigmas may fail to produce such exudates. Third, a thick cuticle may be deposited on the stigmas of abnormal pistils, preventing pollen grains from germinating normally on such a heavily cutinized surface, whereas the cuticle on normal stigmas may be thin or even broken in some places at the point that pollen grains land on the stigmas. We speculate that the poor ability of aberrant pistils to capture pollen grains may result in the low pollination success rates of these abnormal pistils, and may eventually lead to the low seed output of L. chinensis. This work describes megasporogenesis, megagametogenesis, and callose deposition during these processes in L. chinensis. Pistils at the pre- and post-pollination stages were also investigated. The results suggest that callose deposition during megasporogenesis plays an important part in the formation of the normal megagametophyte, and abnormal pistils are usually accompanied by an irregular deposition of callose. Most abnormalities took place during the late stages of pistil development. The high percentage of abnormal pistils and their high rate of pollination failure may be the dominant factors in the low seed set of L. chinensis. Our data suggest that environmental conditions may regulate pistil development and influence the production of seed in this species, but further direct evidence is needed to support this. Acknowledgments This work was supported by the Key Project of the Chinese Academy of Sciences (KSCX1-08) and National Science Fund of China for Distinguished Young Scholars ( ). We thank Dr. Arthur Benson and other anonymous botanists for their critical comments on the first draft of this manuscript. We also thank the Inner Mongolian Grassland Ecosystem Research Station for providing field and laboratory facilities. References Aleemullah, M., Haigh, A.M., Holford, P., Anthesis, anther dehiscence, pistil receptivity and fruit development in the Longum group of Capsicum annuum. Aust. J. Exp. Ag. 40, Arbeloa, A., Herrero, M., Development of the ovular structures in peach [Prunus persica (L.) Batsch]. 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