Seasonal and spatial dynamics of allelochemicals in the submersed macrophyte Myriophyllum spicatum L.

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Verh. Internat. Verein. Limnol. 27 1–4 Stuttgart, December 2000

0368-0770/00/0027-01 $ 1.00

Seasonal and spatial dynamics of allelochemicals in the submersed macrophyte Myriophyllum spicatum L.

Elisabeth M. Gross

Introduction

Myriophyllum spicatum L. (or milfoil for short) is a highly competitive submersed macrophyte which can replace other submersed macrophytes and form monospecific stands. Frequently, epiphyte cover and phytoplankton development is low in M. spicatum dominated systems. Milfoil shoots grow fast and tall and tend to form canopies at the water surface depriving underlying photosynthetic organisms of light. M. spicatum exhibited the highest maximum photosynthetic rate (V_max) in comparison with six other submersed macrophytes (MADSEN et al. 1991).

M. spicatum spreads fast by vegetative fragmentation and can tolerate sediment resuspension better than many other submersed macrophytes (JOHNSON et al.

1998). Milfoil contains and releases algicidal phenolic compounds (PLANAS et al. 1981, GROSS et al.

1996), among them the major allelochemical tellimagrandin II (GROSS et al. 1996). Both released and tissue bound polyphenolic compounds from M.

spicatum inhibit algal and cyanobacterial alkaline phosphatase activity (GROSS et al. 1996).

Submersed macrophytes usually start growing in late spring, biomass maxima are reached in July or August while phytoplankton biomass maxima are reached in April/May and late summer (GOULDER

1969). An early production of allelochemicals by submersed macrophytes in spring could prevent light limitation by epiphytes, filamentous algae and phytoplankton.

In many plants, low nitrogen supply can yield high concentrations of phenolic compounds and vice versa (e.g. ARNOLD et al. 1995). Milfoil growth is often N-limited (GERLOFF & KROMBHOLZ 1966).

Previous investigations with axenic cultures of M.

spicatum revealed the influence of light and nitrogen availability on the production of algicidal polyphenols (GROSS unpublished results).

The aim of this study was to evaluate spatio–temporal variations in the production of algicidal hydrolysable polyphenols in M. spicatum. The

amount of total phenolic compounds (TPC), concentrations of tellimagrandin II and the C/N ratio were determined in M. spicatum apical shoot seg- ments from monospecific stands in four shallow ponds. The following hypotheses were tested. (1) Do concentrations of allelochemicals exhibit spatio–temporal variations? (2) Does the concentration of polyphenolic inhibitors depend on the nitrogen availability for M. spicatum?

Material and methods

Four ponds (No. 131, 132, 224 and 240) of the Cornell Experimental Ponds Area (Ithaca, NY, USA) with nearly monospecific stands of Myriophyllum spi- catum were sampled monthly from April to October 1997. Twenty apical shoot tips of 25-cm length were collected randomly with a hook from the middle of the ponds and transferred immediately to the laboratory for further processing. Shoots were rinsed off with tap water, homogenised in liquid nitrogen, lyo- philised and finely ground in a mortar. For C/N measurements, duplicate samples were analysed with a Carlo-Erba C/N analyser Model 1500. For the analysis of polyphenolic compounds, two replicates of 100-mg plant material were extracted for 2 h with 5 mL acetone–water 1:1 (v/v) at room temperature.

Analysis for TPC was performed as described previously (GROSS et al. 1996). A calibration curve was performed with purified tellimagrandin II. The amount of tellimagrandin II in extracts was determined by HPLC separation as described previously (GROSS et al. 1996). In April, additional samples were taken at ponds 131, 132 and 224. The upper 5 cm of the apical shoots were analysed separately from the adjacent lower 20 cm to test whether the apical meristem of M. spicatum contains higher con- centrations of TPC and tellimagrandin II than the lower shoots. In September three different samples from ponds 132 and 224 were taken to account for variations within one pond.

First publ. in: Verhandlungen der Internationalen Vereinigung für Limnologie 27 (2000), pp. 2116-2119

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2 Allochemical interactions in freshwater ecosystems

Results

Seasonal dynamics of TPC concentration varied strongly between locations (Fig. 1). Pond 131 showed no clear trend with slight maxima of TPC in May and July. Pond 132 exhibited a strong maximum in May with concentrations of 110.7 mg TPC g^-1 DW, pond 224 had a maximum in June with 121.3 mg g^–1. Pond 240 started with very low concentrations in April and May (48.3 and 33.3 mg g^–1, respectively).

Pond 224 had, in general, higher TPC concentrations than the other ponds. A paired t-test revealed significant differences in TPC concen-

tration only between ponds 132 and 224 (P = 0.05) and ponds 224 and 240 (P = 0.03).

Spatio–temporal differences in the concentration of tellimagrandin II were more pronounced. Ponds 131 and 132 exhibited strong maxima in May (16.2 and 14.3 mg g^–1 DW, respectively) but then concentrations dropped to almost zero in pond 131 and stayed at low levels in pond 132. In pond 224 tellimagrandin II concentrations reached a maximum in June (22.0 mg g^–1) and then stayed at high levels (11.7–15.3 mg g^–1). Pond 240 started out very low in the season (2.3 and 2.8 mg g^–1 in April and May, respectively) but reached values

131

A M J J A S O Total phenolic content [mg g-1 DW]

0 50 100

132

A M J J A S O

224

A M J J A S O

240

A M J J A S O Total phenolic content -1[mg g DW] 0

50 100

A M J J A S O Tellimagrandin II [mg g-1 DW]

0 5 10 15 20

A M J J A S O

C/N molar ratio

0 10 20 30

A M J J A S O A M J J A S O A M J J A S O

Tellimagrandin II [mg g-1 DW]

0 5 10 15 20

A M J J A S O A M J J A S O A M J J A S O

C/N molar ratio

0 10 20 30

Pond number

Fig. 1. Seasonal patterns for total phenolic compounds (1st row), tellimagrandin II (2nd row) and C/N molar ratio (3rd row) in Myriophyllum spicatum shoots from four ponds (no. 131, 132, 224, 240, vertical columns) at the Cornell Experimental Pond Facilities. Data are presented as means ± 1 S.D. (n = 2) for TPC and tellimagrandin II.

First publ. in: Verhandlungen der Internationalen Vereinigung für Limnologie 27 (2000),

pp. 2116-2119

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E. M. Gross, Allelochemical production in Myriophyllum 3

between 12 and 13 mg g^–1 in July, September and October. Thus, concentrations of this com- pound varied between 0.02 and 2% of tissue dry weight while TPC concentrations ranged from 3 to 12%. Concentrations of tellimagrandin II were significantly higher in pond 224 throughout the season than in all other ponds (paired t-test: 131 and 224: P = 0.02; 132 and 224: P = 0.01; 224 and 240: P = 0.03)

C/N ratios differed only slightly between ponds. Significant differences in C/N ratios were found only between ponds 132 and 224 (paired t-test P = 0.05). A comparison of the apical meristem and the following lower shoots from three ponds (131, 132 and 224) yielded significant higher concentrations of both TPC, tellimagrandin II and percent tellimagrandin II of TPC in the apical zone (Table 1, paired t- test). C/N ratios were lower in the apical tips than in lower stems, but the difference was not significant (Table 1). Differences for TPC, tellimagrandin II and C/N ratio within one pond were generally smaller than those between ponds (Table 2). Axenic cultures of M. spicatum grown in a mineral medium showed a strong correlation between tissue C/N ratio and tellimagrandin II (R² = 0.73, P = 0.0004) but not between C/N ratio and TPC (R² = 0.13, P =

0.65) (G^ROSS unpublished data). The nitrogen status of milfoil shoots from the experimental ponds had no impact on the content of polyphenolic compounds (C/N vs. TPC: R² = 0.003, P = 0.77, C/N vs. tellimagrandin II: R²

= 0.026, P = 0.38).

Discussion

Myriophyllum spicatum produces high concen- trations of polyphenolic compounds throughout the vegetation period. In general, spatio–temporal variations in TPC concentrations were less pronounced than concentrations of tellimagrandin II. In particular ponds 131 and 132, which are located adjacent to each other, exhibited very similar seasonal patterns.

Pond 240 started out with weak milfoil shoots which had been heavily blanketed by filamentous algae in April and May. Light limitation by this thick epiphyte cover could explain the low TPC and tellimagrandin II concentrations. M.

spicatum produces less TPC under low light conditions (GROSS unpublished results). The strong decline of tellimagrandin II in ponds 131 and 132 was caused by strong herbivory of Euhrychiopsis lecontei (Coleoptera). This weevil can severely damage M. spicatum (NEWMAN et

Table 1. Differences in concentrations of total phenolic compounds (TPC) and tellimagrandin II between apical meristem and lower stem parts.

Lower stem Apical tip Paired t-test

P

TPC (mg g^–1) 41.4 ± 4.3 95.2 ± 10.4 0.0002

Tellimagrandin II (mg g^–1) 0.7 ± 0.6 12.9 ± 2.4 0.0006

% Tellimagrandin II on TPC 1.7 ± 1.7 13.9 ± 2.5 0.0030

C/N ratio 17.8 ± 5.1 13.4 ± 2.6 0.1000

Table 2. Differences in concentrations of total phenolic compounds (TPC), tellimagrandin II and C/N atomic ratio between ponds and within one pond. Multiple sampling within one pond was performed at pond 132 and 224. Data for ponds 132 and 224 are means ± 1 S.D. of three different samples.

Pond no. TPC (mg g^–1) Tellimagrandin II (mg g^–1) C/N ratio

131 53.5 0.4 20.9

132 62.4 ± 4.6 2.9 ± 1.7 23.5 ± 2.7

224 79.6 ± 3.1 15.3 ± 2.5 18.3 ± 2.1

240 75.7 12.2 20.7

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4 Allochemical interactions in freshwater ecosystems

al. 1996). Phenolic compounds from allochtho- nous sources (SERRANO & GUISANDE 1990) and macrophyte-derived polyphenolic compounds (P^LANAS et al. 1981, G^ROSS et al. 1996) can inhibit phytoplankton growth. The increased production of such allelochemicals in spring, as observed with milfoil in this study, should inhibit extensive phytoplankton and epiphyte mass development during the early growth of this macrophyte.

Herbivory by invertebrates or other unknown factors may have masked a possible effect of nitrogen status in milfoil shoots on the production of polyphenolic compounds. Milfoil grown in N-deficient medium prevented development of epiphytes and phytoplankton while P-deficient medium did not (F^ITZGERALD 1969). Previous in vitro studies have shown a highly significant effect of C/N tissue ratio on the content of tellimagrandin II in M. spicatum.

This study and previous laboratory experiments indicate that a variety of factors can act on the production of milfoil allelochemicals. Biotic interference from invertebrate herbivores and dense epiphyte covers which can deprive M. spi- catum of light seem to be of equal importance as abiotic factors such as availability of resources. A comparison of laboratory studies and field observations on allelopathy remains difficult. At the same time, transferring experimental studies to field situations is critical to corroborate the importance of allelopathy in interspecies interactions. Further laboratory, mesocosm and field studies are warranted to elucidate interactive processes between abiotic and biotic factors on the production of alle- lochemicals in M. spicatum.

Acknowledgements

The help of SHAWN KROSZNICK and MICHAEL BOLLER

is gratefully acknowledged. ROBERT L. JOHNSOn pro- vided excellent support at the Cornell Experimental Ponds. This study was supported by a grant from Deutsche Forschungsgemeinschaft (DFG Gr 1441/

2-1).

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Author’s address:

E. M. GROSS, Limnologisches Institut, Universität Konstanz, D-78457 Konstanz, Germany.

E-mail: Elisabeth.Gross@uni-konstanz.de First publ. in: Verhandlungen der Internationalen

Vereinigung für Limnologie 27 (2000), pp. 2116-2119