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Clone specific differences in a Phragmites australis stand
Aquatic Botany 64 (1999) 247–260
Clone specific differences in a Phragmites australis stand II. Seasonal development of morphological and physiological characteristics at the natural site and after transplantation Hardy Rolletschek ∗ , Alexandra Rolletschek, Harald Kühl, Johannes-Günter Kohl Humboldt-Universität, Institut für Biologie/AG Ökologie, Unter den Linden 6, 10099 Berlin, Germany
Abstract Two Phragmites clones, growing adjacent to each other but differing conspicuously in stand structure, were investigated in 1996 regarding seasonal change in morphological and physiological parameters. Both clones showed significant differences in shoot morphology (length, diameter, dry weight, leaf area), maximum above-ground (420 versus 152 g DW m−2 ) and below-ground biomass (2.4 versus 1.5 kg DW m−2 ). Furthermore, physiological parameters (N concentration, N and P content per shoot and m2 , content of dissolved amino acids, N translocation rate) varied between clones in their time course. Clone-specific variations in these characteristics were significant even after transplantation to another field site. Overall, these results suggest that the distinct growth forms and levels of productivity of the two Phragmites clones are the result of genotypic variation. It is hypothesized that both clones follow distinct ecophysiological strategies causing their morphotypic differentiation. Implications of genotypic determination of growth forms are discussed in relation to nutrient supply, N limitation, population plasticity, eutrophication and reed regression. ©1999 Elsevier Science B.V. All rights reserved. Keywords: Genotypic variation; Nutrient cycle; Assimilation type; Translocation type; Dissolved amino acids; Dissolved carbohydrates; N limitation; Phragmites australis
∗ Corresponding author. Current addres: Institut für Pflanzengenetik und Kulturpflanzenforschung Corrensstr. 3 D – 06466 Gatersleben. Tel.: +49-039-482-5264; fax: +49-039-482-5138 E-mail address: [email protected] (H. Rolletschek)
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Fig. 1. View on two monoclonal stands of P. australis growing at the south shore of the eutrophic Lake Seddinsee (Berlin, Germany): Seddin2 (left) and Seddin3 (right).
1. Introduction In general, the productivity of stands of common reed (Phragmites australis (Cav.) Trin. ex Steudel) reflects the trophic state of the site inhabited (Rodewald-Rudescu, 1974). The influence of site conditions on reed development and growth dynamics is well documented (Bornkamm and Raghi-Atri, 1986; Daniels, 1991; Young et al., 1991; Kohl et al., 1998). In adaptation to site conditions, especially nutrient availability in littoral sediments, ecotypes of P. australis are established by selection of genotypes (Björk, 1967; Van der Toorn, 1972; Dykyjova and Hradecka, 1976). However, the question arises which morphological, anatomical and physiological characteristics are of selective advantage for the specific ecotypes. In this study, two stands of P. australis were investigated growing in direct vicinity at the same lakeside, but differing conspicuously in their habitus and stand structure (Fig. 1). In a preliminary study, it was found that both stands are genetically different and their clone-specific morphological variations are evident on a long-time scale (part I. Kühl et al., 1999 this issue). These variations could not be traced back to distinct site conditions. This study focused on the seasonal development of morphological and physiological parameters at the natural site and an experimental field site after transplantation of both clones, aiming: (1) to find evidence that the distinct growth forms are the result of genotypic variation, and (2) to identify physiological factors which are responsible for the development of the two morphotypes.
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2. Materials and methods 2.1. Sampling and analysis of plant material from the natural site The Phragmites clones Seddin2 and Seddin3 were investigated at their natural site in 1996. A detailed description of the site and the reed stand, the sampling scheme and morphometric analyses is given in part I of this study. In the laboratory, the monthly sampled plants (n = 10) were separated into the apex and different internodes (also comprising the respective leaf sheaths and blades). The internodes were enumerated beginning at the uppermost, already elongating, internode. The apex the 2nd, 4th, 8th and the basal internode above ground were taken for analyses. The respective internodes of plants from every clone were combined to one sample and weighed (fresh weight). After drying at 60◦ C to weight constancy, their dry weight (DW) was determined. Their tissue N concentration was analyzed by a CHN analyser (Heraeus, Germany). Their tissue P concentration was measured photometrically (820 nm) after ignition (550◦ C) and acidic extraction according to the methylene-blue method (Murphey and Riley, 1962) using a flow-injection analyzer (Eppendorf, Germany). The dependence of fresh weight, dry weight, and N concentration of the different internodes within culms was described by polynomial regression functions (R2 ranged between 0.92 and 0.99). These functions served for calculating dry weight, the N concentration and the absolute N content of the shoot (Nrel and Nabs ). The same procedure was used for calculation of P concentration and absolute P content of the shoot (Prel and Pabs ). Below-ground biomass was sampled in June and November using a stainless steel corer (17 cm inner diameter). Six cores per stand with a maximum length of 1 m were sliced: the upper 20 cm of soil layer was divided into 10 cm-slices, the lower layer (20–80 cm) into 20 cm-slices. The living rhizomes and roots were removed, washed free of all sediment and dried at 60◦ C. Standing stocks of N and P for roots and rhizomes were calculated from the biomass per sq. meter and their mean Nrel and Prel . 2.2. Internode sampling and analysis of amino acids and carbohydrates Up to five basal culm internodes of shoots were sampled monthly in 1996, washed with distilled water and immediately frozen in liquid N2 . After freeze-drying, internode samples were pulverized under liquid N2 (Mikro-Dismembrator; Braun Biotech, Germany). For determination of dissolved carbohydrates and amino acids, up to 200 mg of powdered samples were extracted three times with 80% ethanol at room temperature, centrifuged (5000g) and the combined supernatants were stored at −20◦ C for subsequent analysis. As an internal standard used for recovery checks, norleucine was added to the samples during extraction. Amino acids in the ethanolic extract were filtered, derivatized with phenylisothiocyanate and measured using a HPLC system (Waters: gradient pump module 600E, diodenarray detector 991, Pico-Tag® method). Dissolved carbohydrates were determined in the same ethanolic extracts using a HPLC system (Dionex DX-100: separation on CarboPac® PA1 columns, pulsed amperometric detector). Standard mixtures of amino acids and carbohydrates were used for identification and quantification of the samples. The totals of dissolved
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amino acids (TotAA) and carbohydrates (TotCARB) were calculated as the sum of all 20 detected and quantified amino acids and three detected carbohydrates (sucrose, glucose, fructose), respectively. Their contents are given in absolute (mol g−1 DM) and relative (% of TotAA and TotCARB) units. 2.3. Transplantation experiment, sampling and analysis of plant material After meristematic reproduction of plant material from Seddin2 and Seddin3 (Tinplant GmbH, Germany), six-week-old plants (referred to as Trans2 and Trans3) were transplanted to the north shore of Lake Seddinsee in the spring of 1995. The experimental habitat consisted of homogeneous, medium-grained sand (0.2–0.5 mm). It was elevated ca. 10 cm above the mean water level, and was not flooded at any time of the year. The experimental plots covered an area of 30 m2 with an initial plant density of 15 plant pots per sq. meter. Porewater concentrations of inorganic nutrients were comparable at both experimental plots but lower compared to the natural site, suggesting lower nutrient availability (Zemlin, pers. commun.). The established Phragmites clones Trans2 and Trans3 were sampled in 1997 and analyzed for morphometric parameters, dry weight, N concentration and N content of the shoot as described above. 2.4. Statistical analyses Means of all parameters were compared using the nonparametric WELCH-test, and the WILCOXON-rank test, if observations were paired. The level of significance was set to P ≤ 0.05. Spearman rank correlation coefficients (rs ) were calculated using a significance level of P ≤ 0.01.
3. Results 3.1. Seasonal development of morphometric parameters 3.1.1. Above-ground biomass Seasonal biomass development of the natural and the experimental Phragmites stands followed similar patterns. Tiller outgrowth, shoot emergence and elongation from April to August were connected with increasing length and leaf area of shoots. Maximum standing crop was reached in August in the natural stands and in September in the experimental stands (Tables 1 and 2). Grazing by Fulica atra and Ondathra zibethicus, and mechanical damage due to wave action caused a decrease in mean shoot length, shoot density and standing crop of Seddin2 and Seddin3 in comparison with the former years (part I), but especially from August to October 1996. The shoots of Seddin3 had significantly higher length, diameter, leaf area and dry weight compared to Seddin2 throughout the year (rank test; Table 1). The same relationships were found at the experimental site for Trans3 and Trans2 (Table 2). These plants passed
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through a developmental stage with increasing shoot length, culm diameters and biomass from 1995 to 1997. All morphometric parameters are expected to increase within the next vegetation periods. The established stands Trans2 and especially Trans3 already reached higher standing crops than at the natural site. This fact was primarily due to the chosen high density of planted pots, rather than to a higher nutrient availability at the experimental site. 3.1.2. Below-ground biomass Differences in the above-ground biomass of the natural reed stands were also reflected in their rhizome and root biomass. Seddin3 reached considerable higher below-ground biomasses than Seddin2 (2.4 ± 1.5 kg DW m−2 versus 1.5 ± 0.8 kg DW m−2 ; pooled data of June and November). Combining these data with maximum standing crop (Table 1), Seddin3 had a higher above-ground/below-ground ratio than Seddin2 (0.18 versus 0.10). In June, Seddin3 plants possessed relatively more root biomass than Seddin2 (58 versus 45% of total below-ground biomass), whereas in November their share at both stands decreased to 30%. The depth profiles showed a trend of biomass decreasing with depth (Fig. 2). The wide confidence intervals reflect the spatial variability within the stands. About 97 ± 5% of the total living biomass was located at 0–40 cm depth. The comparison of biomass data for June and November suggested a decrease in roots and an increase in rhizomes at the end of the season. However, the mean values were not significantly different because of the high spatial variability within the stands. 3.2. Seasonal nitrogen and phosphorus dynamics in the above-ground biomass 3.2.1. Natural site The shoots of Seddin3 contained significantly more Nabs compared to Seddin2 in their seasonal development (Fig. 3). This fact can be attributed to higher shoot DW throughout the year and higher Nrel at least from April to July. From July to August Nrel decreased continuously. After termination of growth, the decrease of Nabs from its maximum in August to the end of the season expresses real N export to rhizomes. This N translocation amounted to 37 and 32% of Nabs maximum at Seddin2 and Seddin3, respectively. The slightly lower translocation of Seddin3 resulted in N enriched old culms in November compared to Seddin2. The maximum above-ground standing stock of N was significantly higher for Seddin3 than for Seddin2 (9.1 versus 3.7 g N m−2 ). The time course of Pabs content per shoot showed similar dynamics as described for nitrogen, nearly two-fold Pabs throughout the year in shoots of Seddin3 compared to Seddin2, with maximum values in August strongly decreasing to the end of the season (Fig. 3). Stand-specific differences in Pabs were caused by higher DW of Seddin3 shoots. Prel of shoots decreased strongly from May to June, while significant differences between stands did not occur. The decrease of Pabs from its maximum to the end of the season (P translocation) amounted to 65 and 66% of Pabs maximum at Seddin2 and Seddin3, respectively. The maximum above-ground standing stock of P was significantly higher for Seddin3 than for Seddin2 (0.53 versus 0.25 g P m−2 ). 3.2.2. Experimental site The shoots of Trans3 plants reached significantly higher mean Nabs contents than those of Trans2 throughout the season (Fig. 4). This was related to both higher DW (Table 2) and
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Fig. 2. Depth profiles of living below-ground biomass at the natural Phragmites-stands Seddin2 and Seddin3 in June and November 1996; means for root and rhizome biomass and 95%-confidence limits for total biomass are shown.
higher N concentrations of Trans3 shoots compared to those of Trans2. After termination of growth, the decrease of Nabs from September to the end of the season (N translocation) amounted to 61 and 49% of Nabs maximum at Trans2 and Trans3, respectively. The considerably lower N translocation of Trans3 resulted in N enriched old culms compared to Trans2. The maximum above-ground standing stock of N was significantly higher for Seddin3 than for Seddin2 (33.5 versus 7.0 g N m−2 ). 3.3. Nitrogen and phosphorus dynamics in the below-ground biomass In both natural stands Nrel of rhizomes increased significantly from June to November, whereas Nrel of roots was nearly constant (Table 3). Rhizomes of Seddin3 reached remarkably higher Nrel at the end of the season than Seddin2, whereas their tissue P level
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Fig. 3. Seasonal development of tissue concentration and absolute content of nitrogen and phosphorus per shoot at the natural Phragmites-stands Seddin2 and Seddin3 in 1996 (mean values, n = 10).
Fig. 4. Seasonal development of tissue concentration and absolute content of nitrogen per shoot at the established Phragmites-stands Trans2 and Trans3 in 1997 (mean values, n = 10).
was comparable. Standing stocks of N increased for rhizomes due to increasing Nrel and biomass, and decreased for roots due to decreasing biomass. In both stands Prel of rhizomes and roots increased significantly from June to November, while Prel of roots was significantly lower than that of rhizomes (Table 3). Phosphorus was accumulated especially in the rhizome layer 10–20 cm below surface (data not shown). Standing stocks of P increased considerably due to increasing Prel and rhizome biomass. The below-ground standing stocks of N and P were remarkably higher for Seddin3 than for Seddin2 at both sampling dates (Table 3).
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Table 3 Concentration and standing stocks of N and P in living roots and rhizomes at the natural Phragmites-stands Seddin2 and Seddin3 in June and November 1996 (means ± SD; n = 6) Month
Rhizome Seddin2
Seddin3
Seddin2
Seddin3
Nrel (mg N g−1 DW)
June November
0.88 ± 0.12 1.20 ± 0.10a
0.95 ± 0.23 1.73 ± 0.48a
1.26 ± 0.08 1.27 ± 0.12
1.31 ± 0.05 1.30 ± 0.02
Prel (g P g−1 DW)
June November
74 ± 53 1169 ± 123a
53 ± 16 1297 ± 303a
16 ± 14 576 ± 26 a
16 ± 16 488 ± 23a
N (g N m−2 )
June November
6.6 12.8a
13.8 25.5a
9.0 5.5
15.2 8.3a
P (mg P m−2 )
June November
55 1247a
77 1914a
11 249a
19 312a
a
Root
Significantly different vs. the respective value in June.
3.4. Seasonal change of amino acid and carbohydrate content and composition During the outgrowth of tillers and emergence of young shoots from May to June, the TotCARB content decreased considerably at both reed stands (Fig. 5(a) and (c) ). The lowest levels were reached in July (Seddin3) and August (Seddin2), while increasing remarkably afterwards. Sucrose was the main component at both stands with mean shares of 87.8 ± 9.5% TotCARB throughout the season except July and November when it decreased to values lower than 45% TotCARB in favour of glucose and fructose. Interestingly, seasonal change in the TotAA content and the TotAA/TotCARB-ratio showed some stand-specific variations. At Seddin2, TotAA was nearly constant between May and August, but dropped significantly in September and October to increase again in November (Fig. 5(a)). At Seddin3, TotAA increased significantly from May to July, but afterwards it dropped continuously till October. In November, it increased again as shown for Seddin2 (Fig. 5(c)). TotAA/TotCARB ratio at Seddin3 showed a strong decrease from July to October. The inverse relationship of both parameters was highly significant for Seddin3 (rs = −0.82), but not for Seddin2. Amino acid composition was similar for both stands. Only seven amino acids amounted to more than 5% of TotAA at any sampling date. These are shown separately in Fig. 5(b) and (d) as stacked columns, while the remaining 13 amino acids were pooled (cumulative to 100%). Asn was the predominant amino acid with a mean share of 56.2 ± 15.3% TotAA (pooled data of Seddin2 and Seddin3 from May to October). Only in November and May (Seddin3) the share of Asn decreased below 11 and 24% TotAA (Seddin2 and Seddin3, respectively). In November, the total content of Ala and Ser was remarkably increased and contributed to 57.0 ± 5.8% and 36.3 ± 6.7% to the TotAA-pool (Seddin2 and Seddin3, respectively). 4. Discussion The natural Phragmites stands investigated in this study differed conspicuously in their habitus, shoot morphology, above-ground and below-ground productivity. These differences
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Fig. 5. Seasonal change in the content and composition of total free amino acids and in the content of total free carbohydrates in basal culm internodes of shoots at the natural Phragmites-stands Seddin2 (a and b) and Seddin3 (c and d) in 1996.
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Fig. 5. (Continued)
were evident in both their seasonal development and comparison over several years (see part I). Furthermore, the seasonal change of physiological parameters (Nrel , Nabs , Pabs , TotAA) showed distinct patterns. At this stage, the question arises which factors are responsible for the clone-specific variations. As essential site conditions like nutrient supply, sediments, flooding, light, shore morphology etc. were equal as already shown in part I of this study, differences in growth conditions may be ruled out, making genotypic variations the likely mechanism of causation. To test the validity of this assumption, both stands were analyzed using genomic fingerprinting. They were shown to be monoclonal and genetically different from each other (see part I). Additionally, both clones were transplanted to another lakeside where they were growing under identical conditions. This experimental site was not flooded and revealed a lower nutrient availability (Zemlin, pers. commun.). Even under these growth conditions differing from the natural site, the clone-specific variations in morphological and physiological parameters appeared. Therefore, it is reasonable to suggest that the distinct growth forms and levels of productivity of the two Phragmites clones are the result of genotypic variation. This assumption is also supported by observations of other authors (Björk, 1967; Van der Toorn, 1972; Dykyjova and Hradecka, 1976). However, unequivocal evidence can probably be obtained only by reciprocal transplantation experiments. Since such experiments have not yet been done, the final decision is still pending. The question remains which probably genetically determined, physiological factors are responsible for the development of the two morphotypes. Rodewald-Rudescu (1974) pointed out that morphometry of P. australis is related to its ploidy level. However, this was checked, and plant samples of both clones had the same (tetraploid) level (Clevering, pers. commun.). Interestingly, the seasonal N dynamics indicate some clonal differences appearing at both the natural and the experimental site. Therefore, we hypothesize that both clones follow distinct ecophysiological strategies causing their morphotypic differentiation. These strategies were previously described as assimilation type and translocation type (Kühl et al.,
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1997). The assimilation type is characterized by longer tillers/young shoots which emerge earlier and, therefore, an elongated autotrophic growth period, a higher tissue level and absolute content of limiting nutrients (N or P) in shoots due to a higher nutrient allocation to shoots and a higher assimilation potential of roots, higher nutrient concentrations in standing old culms at the end of the season due to lower translocation rates, a higher above-ground/below-ground biomass ratio, higher standing stocks of N and P, and relatively little root biomass. Consequently, the assimilation type is a highly-productive Phragmites stand with a pronounced external nutrient cycle (Lippert et al., 1999 in press). Nearly all these characteristics were found for Seddin3. Seddin2 would represent the translocation type with contrasting patterns: (1) The tissue N level of Seddin3 (Trans3) shoots exceeded that of Seddin2 (Trans2) throughout the season at the experimental site and from spring till July at the natural site, respectively. The decline in the Nrel of Seddin3 below that of Seddin2 from July to August is regarded as a deviation from normal behaviour presumably due to grazing. The compensatory production of secondary shoots leads to the assimilation and translocation of additional N, resulting in higher N concentrations in these shoots. However, when the N availability is limited and Seddin3 shoots have a higher N demand for growth (higher dry weight and tissue level), Seddin3 plants are subject to N limited growth. Hence, they are unable to increase their N concentration to the normally reached level. The likely N limitation of Seddin3 is clearly supported by the TotAA/TotCARB ratio as discussed below. (2) The higher Nrel level and Nabs (Pabs ) content of Seddin3 (Trans3) shoots indicate a higher N (P) assimilation and allocation to shoots. This N (P) has to be supplied by additional uptake from the porewater of sediments and/or by their storage pools. At least in the growing season, Seddin3 plants possessed more root biomass than Seddin2 enhancing the assimilation potential. Furthermore, Seddin3 plants revealed a higher Nrel in their rhizomes. Both factors may provide additional nitrogen. (3) The rates of N translocation to the rhizomes were found to be lower in both Seddin3 and Trans3 compared to Seddin2 and Trans2, respectively. (4) This led to N enriched standing old culms at Seddin3 and Trans3 at the end of the season. (5) Seddin3 had a higher above-ground/below-ground biomass ratio than Seddin2. (6) Seddin3 had higher below-ground and above-ground standing stocks of N and P than Seddin2 due to higher concentrations and/or biomasses. Overall, a subdivision of both clones into the assimilation and the translocation type, respectively, seems to be valid. If both Phragmites types are also a common feature of reed stands in different environments will remain open and has to be studied in future. The genotypic determination of growth assumed in this study has important ecological implications: (1) The generally assumed relationship between productivity and trophic state of the site inhabited (Dykyjova and Hradecka, 1976) may be modified by genotypic differences. That means high and low productive morphotypes of P. australis following distinct ecophysiological strategies can be found at similar nutrient levels. Similar considerations have to be made for the relationship between root biomass and nutrient supply (Iwasawa and Roughgarden, 1984; Boar et al., 1989; Marschner et al., 1996). (2) There probably exist different threshold values for nutrient limitation depending on clone-specific demand for growth. The TotAA/TotCARB ratio was previously shown as a significant indicator of the N balance of plants (Kohl et al., 1998). When N supply is limited, the amino acid pool decreases while the pool of photosynthates (dissolved carbohydrates) increases. Hence, the strongly decreasing TotAA/TotCARB ratio at Seddin3 beginning from July points to N
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limited growth due to grazing. At Seddin2, this ratio increased later and to a lower extent. Despite the similar N availability for both stands (see part I), the clone with the higher N demand (Seddin3) reached the N limited growth phase earlier. Thus, it can be concluded that the treshold values were different. (3) The high population plasticity in performance and growth of plants like P. australis is ensured by the simultaneous occurrence of clones with distinct ecophysiological strategies. Very probably, however, any single clone has a much lower plasticity. This implies that (monoclonal) reed stands adapted to low nutrient levels may decline if nutrient supply strongly increases. In this case eutrophication acts directly as a die-back factor for P. australis, although ecotypes exist which are obviously well-adapted to hypertrophic conditions (Brix, 1994; Tanner, 1996). Besides this, the clones probably also differ in their susceptibility to indirect effects of eutrophication, such as litter accumulation and decomposition (Armstrong et al., 1996; Van der Putten et al., 1997), or other die-back factors. For example, both Seddin2 and Seddin3 were subject to similar levels of grazing damage and shoot loss. Nevertheless, the hypoxic stress in basal and below-ground plant parts indicated by the high fraction of Ala and Ser in culm internodes (Rolletschek et al., 1998, and 1999 this volume) was much higher for Seddin2. If there exists any relationship between both facts, Seddin2 seems to be more susceptible to this environmental factor.
Acknowledgements This study was financed by the Environment and Climate Programme of the European Commission (contract No. ENV4-CT95-0147: EUREED). The plantations were performed by the Senatsverwaltung für Stadtentwicklung, Umweltschutz und Technologie (Berlin). The data on ploidy level were kindly provided by Dr. O. Clevering, Netherlands Institute of Ecology. Furthermore, R. Kräft, G. König and R. Zemlin are gratefully acknowledged for excellent technical assistance. The authors thank the anonymous reviewers for constructive criticism. References Armstrong, J., Armstrong, W., van der Putten, W.H., 1996. Phragmites die-back: bud and root death, blockages within the aeration and vascular systems and the possible role of phytotoxins. New Phytol. 133, 399–414. Björk, S., 1967. Ecologic investigations of Phragmites communis. Studies in theoretic and applied limnology. Folia Limnol. Scand. 14, 1–248. Boar, R.R., Crook, C.E., Moss, B., 1989. Regression of Phragmites australis reedswamps and recent changes of water chemistry in the Norfolk Broadland, England. Aquat. Bot. 35, 41–55. Bornkamm, R., Raghi-Atri, F., 1986. Über die Wirkung unterschiedlicher Gaben von Stickstoff und Phosphor auf die Entwicklung von Phragmites australis (Cav.) Trin. ex Steudel. Arch. Hydrobiol. 105, 423–441. Brix, H., 1994. Constructed wetlands for municipal wastewater treatment in Europe. In: Mitsch, W.J. (Ed.), Global Wetlands: Old World and New. Elsevier, Amsterdam, The Netherlands, pp. 325–333. Daniels, R.E., 1991. Variation in performance of Phragmites australis in experimental culture. Aquat. Bot. 42, 41–48. Dykyjova, D., Hradecka, D., 1976. Production ecology of Phragmites communis. 1. Relations of two ecotypes to the microclimate and nutrient conditions of habitat. Folia Geobot. Phytotax. 11, 225–259. Iwasawa, Y., Roughgarden, J., 1984. Shoot/root balance of plants: optimal growth of a system with many vegetative organs. Theor. Pop. Biol. 25, 78–104.
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Kohl, J.-G., Woitke, P., Kühl, H., Dewender, M., König, G., 1998. Seasonal changes in dissolved amino acids and sugars in basal culm internodes as physiological indicators of the C/N-balance of Phragmites australis at littoral sites of different trophic status. Aquat. Bot. 60, 221–240. Kühl, H., Woitke, P., Kohl, J.-G., 1997. Strategies of nitrogen cycling of Phragmites australis at two sites differing in nutrient availability. Int. Revue Ges Hydrobiol. 82, 57–66. Kühl, H., Koppitz, H., Rolletschek, H., Kohl, J.-G., 1999. Clone specific differences in a Phragmites australis stand I. Morphology, genetics and site description. Aquat. Bot. 64, 235–246. Lippert, I., Rolletschek, H., Kühl, H., Kohl, J.-G., 1999. Internal and external nutrient cycles in stands of Phragmites australis — a model for two ecotypes, Hydrobiology, in press. Marschner, H., Kirkby, E.A., Cakmak, I., 1996. Effect of mineral nutritional status on shoot-root partitioning of photoassimilates and cycling of mineral nutrients. J. Exp. Bot. 47, 1255–1263. Murphey, J., Riley, J.P., 1962. A modified single solution method for determination of phosphate in natural waters. Ann. Chim. Acta 27, 31–36. Rodewald-Rudescu, L., 1974. Das Schilfrohr Phragmites australis. E. Schweizerbart‘sche Verlagsbuchhandlung, Stuttgart, 302 pp. Rolletschek, H., Rolletschek, A., Henze, R., Kohl, J.-G., 1998. Implications of missing efflux sites on convective ventilation and amino acid metabolism in Phragmites australis. New Phytol. 140, 121–127. Rolletschek, H., Hartzendorf, T., Rolletschek, A., Kohl, J.-G., 1999. Biometric variation of Phragmites australis affecting convective ventilation and amino acid metabolism. Aquat. Bot. 64, 291–302. Tanner, C.C., 1996. Plants for constructed wetland treatment systems — a comparison of the growth and nutrient uptake of eight emergent species. Ecol. Eng. 7, 59–83. Van der Putten, W.H., Peters, B.A.M., Vandenberg, A.S., 1997. Effects of litter on substrate conditions and growth of emergent macrophytes. New Phytol. 135, 527–537. Van der Toorn, J. , 1972. Variability of Phragmites communis in relation to the environment. Van Zee Tot Land 48, 1–122. Young, S.W., Davies, D.H., Milligan, P.J., 1991. The potential of anatomical features of the common reed Phragmites australis (Cav.) Trin. ex Steudel as a biotic indicator of adjoining land use. Arch. Hydrobiol. 122, 297–304.
Clone specific differences in a Phragmites australis stand II. Seasonal development of morphological and physiological characteristics at the natural site and after transplantation Hardy Rolletschek ∗ , Alexandra Rolletschek, Harald Kühl, Johannes-Günter Kohl Humboldt-Universität, Institut für Biologie/AG Ökologie, Unter den Linden 6, 10099 Berlin, Germany
Abstract Two Phragmites clones, growing adjacent to each other but differing conspicuously in stand structure, were investigated in 1996 regarding seasonal change in morphological and physiological parameters. Both clones showed significant differences in shoot morphology (length, diameter, dry weight, leaf area), maximum above-ground (420 versus 152 g DW m−2 ) and below-ground biomass (2.4 versus 1.5 kg DW m−2 ). Furthermore, physiological parameters (N concentration, N and P content per shoot and m2 , content of dissolved amino acids, N translocation rate) varied between clones in their time course. Clone-specific variations in these characteristics were significant even after transplantation to another field site. Overall, these results suggest that the distinct growth forms and levels of productivity of the two Phragmites clones are the result of genotypic variation. It is hypothesized that both clones follow distinct ecophysiological strategies causing their morphotypic differentiation. Implications of genotypic determination of growth forms are discussed in relation to nutrient supply, N limitation, population plasticity, eutrophication and reed regression. ©1999 Elsevier Science B.V. All rights reserved. Keywords: Genotypic variation; Nutrient cycle; Assimilation type; Translocation type; Dissolved amino acids; Dissolved carbohydrates; N limitation; Phragmites australis
∗ Corresponding author. Current addres: Institut für Pflanzengenetik und Kulturpflanzenforschung Corrensstr. 3 D – 06466 Gatersleben. Tel.: +49-039-482-5264; fax: +49-039-482-5138 E-mail address: [email protected] (H. Rolletschek)
0304-3770/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 7 7 0 ( 9 9 ) 0 0 0 5 4 - 6
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Fig. 1. View on two monoclonal stands of P. australis growing at the south shore of the eutrophic Lake Seddinsee (Berlin, Germany): Seddin2 (left) and Seddin3 (right).
1. Introduction In general, the productivity of stands of common reed (Phragmites australis (Cav.) Trin. ex Steudel) reflects the trophic state of the site inhabited (Rodewald-Rudescu, 1974). The influence of site conditions on reed development and growth dynamics is well documented (Bornkamm and Raghi-Atri, 1986; Daniels, 1991; Young et al., 1991; Kohl et al., 1998). In adaptation to site conditions, especially nutrient availability in littoral sediments, ecotypes of P. australis are established by selection of genotypes (Björk, 1967; Van der Toorn, 1972; Dykyjova and Hradecka, 1976). However, the question arises which morphological, anatomical and physiological characteristics are of selective advantage for the specific ecotypes. In this study, two stands of P. australis were investigated growing in direct vicinity at the same lakeside, but differing conspicuously in their habitus and stand structure (Fig. 1). In a preliminary study, it was found that both stands are genetically different and their clone-specific morphological variations are evident on a long-time scale (part I. Kühl et al., 1999 this issue). These variations could not be traced back to distinct site conditions. This study focused on the seasonal development of morphological and physiological parameters at the natural site and an experimental field site after transplantation of both clones, aiming: (1) to find evidence that the distinct growth forms are the result of genotypic variation, and (2) to identify physiological factors which are responsible for the development of the two morphotypes.
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2. Materials and methods 2.1. Sampling and analysis of plant material from the natural site The Phragmites clones Seddin2 and Seddin3 were investigated at their natural site in 1996. A detailed description of the site and the reed stand, the sampling scheme and morphometric analyses is given in part I of this study. In the laboratory, the monthly sampled plants (n = 10) were separated into the apex and different internodes (also comprising the respective leaf sheaths and blades). The internodes were enumerated beginning at the uppermost, already elongating, internode. The apex the 2nd, 4th, 8th and the basal internode above ground were taken for analyses. The respective internodes of plants from every clone were combined to one sample and weighed (fresh weight). After drying at 60◦ C to weight constancy, their dry weight (DW) was determined. Their tissue N concentration was analyzed by a CHN analyser (Heraeus, Germany). Their tissue P concentration was measured photometrically (820 nm) after ignition (550◦ C) and acidic extraction according to the methylene-blue method (Murphey and Riley, 1962) using a flow-injection analyzer (Eppendorf, Germany). The dependence of fresh weight, dry weight, and N concentration of the different internodes within culms was described by polynomial regression functions (R2 ranged between 0.92 and 0.99). These functions served for calculating dry weight, the N concentration and the absolute N content of the shoot (Nrel and Nabs ). The same procedure was used for calculation of P concentration and absolute P content of the shoot (Prel and Pabs ). Below-ground biomass was sampled in June and November using a stainless steel corer (17 cm inner diameter). Six cores per stand with a maximum length of 1 m were sliced: the upper 20 cm of soil layer was divided into 10 cm-slices, the lower layer (20–80 cm) into 20 cm-slices. The living rhizomes and roots were removed, washed free of all sediment and dried at 60◦ C. Standing stocks of N and P for roots and rhizomes were calculated from the biomass per sq. meter and their mean Nrel and Prel . 2.2. Internode sampling and analysis of amino acids and carbohydrates Up to five basal culm internodes of shoots were sampled monthly in 1996, washed with distilled water and immediately frozen in liquid N2 . After freeze-drying, internode samples were pulverized under liquid N2 (Mikro-Dismembrator; Braun Biotech, Germany). For determination of dissolved carbohydrates and amino acids, up to 200 mg of powdered samples were extracted three times with 80% ethanol at room temperature, centrifuged (5000g) and the combined supernatants were stored at −20◦ C for subsequent analysis. As an internal standard used for recovery checks, norleucine was added to the samples during extraction. Amino acids in the ethanolic extract were filtered, derivatized with phenylisothiocyanate and measured using a HPLC system (Waters: gradient pump module 600E, diodenarray detector 991, Pico-Tag® method). Dissolved carbohydrates were determined in the same ethanolic extracts using a HPLC system (Dionex DX-100: separation on CarboPac® PA1 columns, pulsed amperometric detector). Standard mixtures of amino acids and carbohydrates were used for identification and quantification of the samples. The totals of dissolved
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amino acids (TotAA) and carbohydrates (TotCARB) were calculated as the sum of all 20 detected and quantified amino acids and three detected carbohydrates (sucrose, glucose, fructose), respectively. Their contents are given in absolute (mol g−1 DM) and relative (% of TotAA and TotCARB) units. 2.3. Transplantation experiment, sampling and analysis of plant material After meristematic reproduction of plant material from Seddin2 and Seddin3 (Tinplant GmbH, Germany), six-week-old plants (referred to as Trans2 and Trans3) were transplanted to the north shore of Lake Seddinsee in the spring of 1995. The experimental habitat consisted of homogeneous, medium-grained sand (0.2–0.5 mm). It was elevated ca. 10 cm above the mean water level, and was not flooded at any time of the year. The experimental plots covered an area of 30 m2 with an initial plant density of 15 plant pots per sq. meter. Porewater concentrations of inorganic nutrients were comparable at both experimental plots but lower compared to the natural site, suggesting lower nutrient availability (Zemlin, pers. commun.). The established Phragmites clones Trans2 and Trans3 were sampled in 1997 and analyzed for morphometric parameters, dry weight, N concentration and N content of the shoot as described above. 2.4. Statistical analyses Means of all parameters were compared using the nonparametric WELCH-test, and the WILCOXON-rank test, if observations were paired. The level of significance was set to P ≤ 0.05. Spearman rank correlation coefficients (rs ) were calculated using a significance level of P ≤ 0.01.
3. Results 3.1. Seasonal development of morphometric parameters 3.1.1. Above-ground biomass Seasonal biomass development of the natural and the experimental Phragmites stands followed similar patterns. Tiller outgrowth, shoot emergence and elongation from April to August were connected with increasing length and leaf area of shoots. Maximum standing crop was reached in August in the natural stands and in September in the experimental stands (Tables 1 and 2). Grazing by Fulica atra and Ondathra zibethicus, and mechanical damage due to wave action caused a decrease in mean shoot length, shoot density and standing crop of Seddin2 and Seddin3 in comparison with the former years (part I), but especially from August to October 1996. The shoots of Seddin3 had significantly higher length, diameter, leaf area and dry weight compared to Seddin2 throughout the year (rank test; Table 1). The same relationships were found at the experimental site for Trans3 and Trans2 (Table 2). These plants passed
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through a developmental stage with increasing shoot length, culm diameters and biomass from 1995 to 1997. All morphometric parameters are expected to increase within the next vegetation periods. The established stands Trans2 and especially Trans3 already reached higher standing crops than at the natural site. This fact was primarily due to the chosen high density of planted pots, rather than to a higher nutrient availability at the experimental site. 3.1.2. Below-ground biomass Differences in the above-ground biomass of the natural reed stands were also reflected in their rhizome and root biomass. Seddin3 reached considerable higher below-ground biomasses than Seddin2 (2.4 ± 1.5 kg DW m−2 versus 1.5 ± 0.8 kg DW m−2 ; pooled data of June and November). Combining these data with maximum standing crop (Table 1), Seddin3 had a higher above-ground/below-ground ratio than Seddin2 (0.18 versus 0.10). In June, Seddin3 plants possessed relatively more root biomass than Seddin2 (58 versus 45% of total below-ground biomass), whereas in November their share at both stands decreased to 30%. The depth profiles showed a trend of biomass decreasing with depth (Fig. 2). The wide confidence intervals reflect the spatial variability within the stands. About 97 ± 5% of the total living biomass was located at 0–40 cm depth. The comparison of biomass data for June and November suggested a decrease in roots and an increase in rhizomes at the end of the season. However, the mean values were not significantly different because of the high spatial variability within the stands. 3.2. Seasonal nitrogen and phosphorus dynamics in the above-ground biomass 3.2.1. Natural site The shoots of Seddin3 contained significantly more Nabs compared to Seddin2 in their seasonal development (Fig. 3). This fact can be attributed to higher shoot DW throughout the year and higher Nrel at least from April to July. From July to August Nrel decreased continuously. After termination of growth, the decrease of Nabs from its maximum in August to the end of the season expresses real N export to rhizomes. This N translocation amounted to 37 and 32% of Nabs maximum at Seddin2 and Seddin3, respectively. The slightly lower translocation of Seddin3 resulted in N enriched old culms in November compared to Seddin2. The maximum above-ground standing stock of N was significantly higher for Seddin3 than for Seddin2 (9.1 versus 3.7 g N m−2 ). The time course of Pabs content per shoot showed similar dynamics as described for nitrogen, nearly two-fold Pabs throughout the year in shoots of Seddin3 compared to Seddin2, with maximum values in August strongly decreasing to the end of the season (Fig. 3). Stand-specific differences in Pabs were caused by higher DW of Seddin3 shoots. Prel of shoots decreased strongly from May to June, while significant differences between stands did not occur. The decrease of Pabs from its maximum to the end of the season (P translocation) amounted to 65 and 66% of Pabs maximum at Seddin2 and Seddin3, respectively. The maximum above-ground standing stock of P was significantly higher for Seddin3 than for Seddin2 (0.53 versus 0.25 g P m−2 ). 3.2.2. Experimental site The shoots of Trans3 plants reached significantly higher mean Nabs contents than those of Trans2 throughout the season (Fig. 4). This was related to both higher DW (Table 2) and
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Fig. 2. Depth profiles of living below-ground biomass at the natural Phragmites-stands Seddin2 and Seddin3 in June and November 1996; means for root and rhizome biomass and 95%-confidence limits for total biomass are shown.
higher N concentrations of Trans3 shoots compared to those of Trans2. After termination of growth, the decrease of Nabs from September to the end of the season (N translocation) amounted to 61 and 49% of Nabs maximum at Trans2 and Trans3, respectively. The considerably lower N translocation of Trans3 resulted in N enriched old culms compared to Trans2. The maximum above-ground standing stock of N was significantly higher for Seddin3 than for Seddin2 (33.5 versus 7.0 g N m−2 ). 3.3. Nitrogen and phosphorus dynamics in the below-ground biomass In both natural stands Nrel of rhizomes increased significantly from June to November, whereas Nrel of roots was nearly constant (Table 3). Rhizomes of Seddin3 reached remarkably higher Nrel at the end of the season than Seddin2, whereas their tissue P level
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Fig. 3. Seasonal development of tissue concentration and absolute content of nitrogen and phosphorus per shoot at the natural Phragmites-stands Seddin2 and Seddin3 in 1996 (mean values, n = 10).
Fig. 4. Seasonal development of tissue concentration and absolute content of nitrogen per shoot at the established Phragmites-stands Trans2 and Trans3 in 1997 (mean values, n = 10).
was comparable. Standing stocks of N increased for rhizomes due to increasing Nrel and biomass, and decreased for roots due to decreasing biomass. In both stands Prel of rhizomes and roots increased significantly from June to November, while Prel of roots was significantly lower than that of rhizomes (Table 3). Phosphorus was accumulated especially in the rhizome layer 10–20 cm below surface (data not shown). Standing stocks of P increased considerably due to increasing Prel and rhizome biomass. The below-ground standing stocks of N and P were remarkably higher for Seddin3 than for Seddin2 at both sampling dates (Table 3).
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Table 3 Concentration and standing stocks of N and P in living roots and rhizomes at the natural Phragmites-stands Seddin2 and Seddin3 in June and November 1996 (means ± SD; n = 6) Month
Rhizome Seddin2
Seddin3
Seddin2
Seddin3
Nrel (mg N g−1 DW)
June November
0.88 ± 0.12 1.20 ± 0.10a
0.95 ± 0.23 1.73 ± 0.48a
1.26 ± 0.08 1.27 ± 0.12
1.31 ± 0.05 1.30 ± 0.02
Prel (g P g−1 DW)
June November
74 ± 53 1169 ± 123a
53 ± 16 1297 ± 303a
16 ± 14 576 ± 26 a
16 ± 16 488 ± 23a
N (g N m−2 )
June November
6.6 12.8a
13.8 25.5a
9.0 5.5
15.2 8.3a
P (mg P m−2 )
June November
55 1247a
77 1914a
11 249a
19 312a
a
Root
Significantly different vs. the respective value in June.
3.4. Seasonal change of amino acid and carbohydrate content and composition During the outgrowth of tillers and emergence of young shoots from May to June, the TotCARB content decreased considerably at both reed stands (Fig. 5(a) and (c) ). The lowest levels were reached in July (Seddin3) and August (Seddin2), while increasing remarkably afterwards. Sucrose was the main component at both stands with mean shares of 87.8 ± 9.5% TotCARB throughout the season except July and November when it decreased to values lower than 45% TotCARB in favour of glucose and fructose. Interestingly, seasonal change in the TotAA content and the TotAA/TotCARB-ratio showed some stand-specific variations. At Seddin2, TotAA was nearly constant between May and August, but dropped significantly in September and October to increase again in November (Fig. 5(a)). At Seddin3, TotAA increased significantly from May to July, but afterwards it dropped continuously till October. In November, it increased again as shown for Seddin2 (Fig. 5(c)). TotAA/TotCARB ratio at Seddin3 showed a strong decrease from July to October. The inverse relationship of both parameters was highly significant for Seddin3 (rs = −0.82), but not for Seddin2. Amino acid composition was similar for both stands. Only seven amino acids amounted to more than 5% of TotAA at any sampling date. These are shown separately in Fig. 5(b) and (d) as stacked columns, while the remaining 13 amino acids were pooled (cumulative to 100%). Asn was the predominant amino acid with a mean share of 56.2 ± 15.3% TotAA (pooled data of Seddin2 and Seddin3 from May to October). Only in November and May (Seddin3) the share of Asn decreased below 11 and 24% TotAA (Seddin2 and Seddin3, respectively). In November, the total content of Ala and Ser was remarkably increased and contributed to 57.0 ± 5.8% and 36.3 ± 6.7% to the TotAA-pool (Seddin2 and Seddin3, respectively). 4. Discussion The natural Phragmites stands investigated in this study differed conspicuously in their habitus, shoot morphology, above-ground and below-ground productivity. These differences
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Fig. 5. Seasonal change in the content and composition of total free amino acids and in the content of total free carbohydrates in basal culm internodes of shoots at the natural Phragmites-stands Seddin2 (a and b) and Seddin3 (c and d) in 1996.
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Fig. 5. (Continued)
were evident in both their seasonal development and comparison over several years (see part I). Furthermore, the seasonal change of physiological parameters (Nrel , Nabs , Pabs , TotAA) showed distinct patterns. At this stage, the question arises which factors are responsible for the clone-specific variations. As essential site conditions like nutrient supply, sediments, flooding, light, shore morphology etc. were equal as already shown in part I of this study, differences in growth conditions may be ruled out, making genotypic variations the likely mechanism of causation. To test the validity of this assumption, both stands were analyzed using genomic fingerprinting. They were shown to be monoclonal and genetically different from each other (see part I). Additionally, both clones were transplanted to another lakeside where they were growing under identical conditions. This experimental site was not flooded and revealed a lower nutrient availability (Zemlin, pers. commun.). Even under these growth conditions differing from the natural site, the clone-specific variations in morphological and physiological parameters appeared. Therefore, it is reasonable to suggest that the distinct growth forms and levels of productivity of the two Phragmites clones are the result of genotypic variation. This assumption is also supported by observations of other authors (Björk, 1967; Van der Toorn, 1972; Dykyjova and Hradecka, 1976). However, unequivocal evidence can probably be obtained only by reciprocal transplantation experiments. Since such experiments have not yet been done, the final decision is still pending. The question remains which probably genetically determined, physiological factors are responsible for the development of the two morphotypes. Rodewald-Rudescu (1974) pointed out that morphometry of P. australis is related to its ploidy level. However, this was checked, and plant samples of both clones had the same (tetraploid) level (Clevering, pers. commun.). Interestingly, the seasonal N dynamics indicate some clonal differences appearing at both the natural and the experimental site. Therefore, we hypothesize that both clones follow distinct ecophysiological strategies causing their morphotypic differentiation. These strategies were previously described as assimilation type and translocation type (Kühl et al.,
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1997). The assimilation type is characterized by longer tillers/young shoots which emerge earlier and, therefore, an elongated autotrophic growth period, a higher tissue level and absolute content of limiting nutrients (N or P) in shoots due to a higher nutrient allocation to shoots and a higher assimilation potential of roots, higher nutrient concentrations in standing old culms at the end of the season due to lower translocation rates, a higher above-ground/below-ground biomass ratio, higher standing stocks of N and P, and relatively little root biomass. Consequently, the assimilation type is a highly-productive Phragmites stand with a pronounced external nutrient cycle (Lippert et al., 1999 in press). Nearly all these characteristics were found for Seddin3. Seddin2 would represent the translocation type with contrasting patterns: (1) The tissue N level of Seddin3 (Trans3) shoots exceeded that of Seddin2 (Trans2) throughout the season at the experimental site and from spring till July at the natural site, respectively. The decline in the Nrel of Seddin3 below that of Seddin2 from July to August is regarded as a deviation from normal behaviour presumably due to grazing. The compensatory production of secondary shoots leads to the assimilation and translocation of additional N, resulting in higher N concentrations in these shoots. However, when the N availability is limited and Seddin3 shoots have a higher N demand for growth (higher dry weight and tissue level), Seddin3 plants are subject to N limited growth. Hence, they are unable to increase their N concentration to the normally reached level. The likely N limitation of Seddin3 is clearly supported by the TotAA/TotCARB ratio as discussed below. (2) The higher Nrel level and Nabs (Pabs ) content of Seddin3 (Trans3) shoots indicate a higher N (P) assimilation and allocation to shoots. This N (P) has to be supplied by additional uptake from the porewater of sediments and/or by their storage pools. At least in the growing season, Seddin3 plants possessed more root biomass than Seddin2 enhancing the assimilation potential. Furthermore, Seddin3 plants revealed a higher Nrel in their rhizomes. Both factors may provide additional nitrogen. (3) The rates of N translocation to the rhizomes were found to be lower in both Seddin3 and Trans3 compared to Seddin2 and Trans2, respectively. (4) This led to N enriched standing old culms at Seddin3 and Trans3 at the end of the season. (5) Seddin3 had a higher above-ground/below-ground biomass ratio than Seddin2. (6) Seddin3 had higher below-ground and above-ground standing stocks of N and P than Seddin2 due to higher concentrations and/or biomasses. Overall, a subdivision of both clones into the assimilation and the translocation type, respectively, seems to be valid. If both Phragmites types are also a common feature of reed stands in different environments will remain open and has to be studied in future. The genotypic determination of growth assumed in this study has important ecological implications: (1) The generally assumed relationship between productivity and trophic state of the site inhabited (Dykyjova and Hradecka, 1976) may be modified by genotypic differences. That means high and low productive morphotypes of P. australis following distinct ecophysiological strategies can be found at similar nutrient levels. Similar considerations have to be made for the relationship between root biomass and nutrient supply (Iwasawa and Roughgarden, 1984; Boar et al., 1989; Marschner et al., 1996). (2) There probably exist different threshold values for nutrient limitation depending on clone-specific demand for growth. The TotAA/TotCARB ratio was previously shown as a significant indicator of the N balance of plants (Kohl et al., 1998). When N supply is limited, the amino acid pool decreases while the pool of photosynthates (dissolved carbohydrates) increases. Hence, the strongly decreasing TotAA/TotCARB ratio at Seddin3 beginning from July points to N
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limited growth due to grazing. At Seddin2, this ratio increased later and to a lower extent. Despite the similar N availability for both stands (see part I), the clone with the higher N demand (Seddin3) reached the N limited growth phase earlier. Thus, it can be concluded that the treshold values were different. (3) The high population plasticity in performance and growth of plants like P. australis is ensured by the simultaneous occurrence of clones with distinct ecophysiological strategies. Very probably, however, any single clone has a much lower plasticity. This implies that (monoclonal) reed stands adapted to low nutrient levels may decline if nutrient supply strongly increases. In this case eutrophication acts directly as a die-back factor for P. australis, although ecotypes exist which are obviously well-adapted to hypertrophic conditions (Brix, 1994; Tanner, 1996). Besides this, the clones probably also differ in their susceptibility to indirect effects of eutrophication, such as litter accumulation and decomposition (Armstrong et al., 1996; Van der Putten et al., 1997), or other die-back factors. For example, both Seddin2 and Seddin3 were subject to similar levels of grazing damage and shoot loss. Nevertheless, the hypoxic stress in basal and below-ground plant parts indicated by the high fraction of Ala and Ser in culm internodes (Rolletschek et al., 1998, and 1999 this volume) was much higher for Seddin2. If there exists any relationship between both facts, Seddin2 seems to be more susceptible to this environmental factor.
Acknowledgements This study was financed by the Environment and Climate Programme of the European Commission (contract No. ENV4-CT95-0147: EUREED). The plantations were performed by the Senatsverwaltung für Stadtentwicklung, Umweltschutz und Technologie (Berlin). The data on ploidy level were kindly provided by Dr. O. Clevering, Netherlands Institute of Ecology. Furthermore, R. Kräft, G. König and R. Zemlin are gratefully acknowledged for excellent technical assistance. The authors thank the anonymous reviewers for constructive criticism. References Armstrong, J., Armstrong, W., van der Putten, W.H., 1996. Phragmites die-back: bud and root death, blockages within the aeration and vascular systems and the possible role of phytotoxins. New Phytol. 133, 399–414. Björk, S., 1967. Ecologic investigations of Phragmites communis. Studies in theoretic and applied limnology. Folia Limnol. Scand. 14, 1–248. Boar, R.R., Crook, C.E., Moss, B., 1989. Regression of Phragmites australis reedswamps and recent changes of water chemistry in the Norfolk Broadland, England. Aquat. Bot. 35, 41–55. Bornkamm, R., Raghi-Atri, F., 1986. Über die Wirkung unterschiedlicher Gaben von Stickstoff und Phosphor auf die Entwicklung von Phragmites australis (Cav.) Trin. ex Steudel. Arch. Hydrobiol. 105, 423–441. Brix, H., 1994. Constructed wetlands for municipal wastewater treatment in Europe. In: Mitsch, W.J. (Ed.), Global Wetlands: Old World and New. Elsevier, Amsterdam, The Netherlands, pp. 325–333. Daniels, R.E., 1991. Variation in performance of Phragmites australis in experimental culture. Aquat. Bot. 42, 41–48. Dykyjova, D., Hradecka, D., 1976. Production ecology of Phragmites communis. 1. Relations of two ecotypes to the microclimate and nutrient conditions of habitat. Folia Geobot. Phytotax. 11, 225–259. Iwasawa, Y., Roughgarden, J., 1984. Shoot/root balance of plants: optimal growth of a system with many vegetative organs. Theor. Pop. Biol. 25, 78–104.
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