Influence of photoperiod on growth, pigment composition and vegetative propagule formation for Potamogeton nodosus Poir. and Potamogeton pectinatus L.

Aquatic Botany, 28 (1987) 103-112 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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INFLUENCE OF PHOTOPERIOD ON GROWTH, PIGMENT COMPOSITION AND VEGETATIVE PROPAGULE FORMATION FOR POTAMOGETON NODOSUS POIR. AND POTAMOGETON PECTINATUS L.

DAVID F. SPENCER and LARS W.J. ANDERSON

USDA-ARS Aquatic Weed Research Laboratory, University of California, Davis, CA 95616 (U.S.A.) (Accepted for publication 9 December 1986)

ABSTRACT

Spencer, D.F. and Anderson, L.W.J., 1987. Influence of photoperiod on growth, pigment composition and vegetative propagule formation for Potamogeton nodosus Poir. and Potamogeton pectinatus L. A quat. Bot., 28:103-112. Photoperiods of 10 or 12 h enhanced vegetative propagule production by Potamogeton nodosus Poir. and P. pectinatus L. The proportion of plants producing vegetative propagules and the number and weight of propagules per plant increased over time, and were greatest after 8 weeks. Maximum vegetative propagule production was 12 ___6 tubers per plant ( mean __S.D.; n = 7) for P. pectinatus and 5 _+3 for P. nodosus. Tuber or winter-bud dry weight accounted for 38 or 27% of total plant dry weight, respectively, for plants grown for 56 days under a 10-h photoperiod. Vegetative propagules appeared to be produced instead of new shoot tissue under short-day conditions. Chlorophyll a and carotenoid content were lower for P. nodosus at short photoperiods, but not for P. pectinatus. The changes in chlorophyll a:b and carotenoid:ratio chlorophyll a suggest that synthesis/degradation of these pigments is tightly coupled and that short photoperiods may initiate senescence in P. nodosus.

INTRODUCTION

V e g e t a t i v e r e p r o d u c t i o n is g e n e r a l l y a m o r e i m p o r t a n t m e a n s o f p e r e n n a t i o n t h a n sexual r e p r o d u c t i o n for m a n y species o f s u b m e r s e d a q u a t i c m a c r o p h y t e s ( S c u l t h o r p e , 1967; H u t c h i n s o n , 1975). A q u a t i c m a c r o p h y t e s p r o d u c e a n u m b e r of m o r p h o l o g i c a l l y d i s t i n c t v e g e t a t i v e p r o p a g u l e s , s u c h as t u b e r s , t u r i o n s a n d w i n t e r b u d s ( H u t c h i n s o n , 1975). I n t e m p e r a t e species, t h e s e s t r u c t u r e s are t y p i c a l l y f o r m e d in t h e a u t u m n . H o w e v e r , t h e role t h a t specific e n v i r o n m e n t a l f a c t o r s p l a y in r e g u l a t i n g t h e f o r m a t i o n of t h e s e s t r u c t u r e s is p o o r l y k n o w n for m o s t species. T h e few species t h a t h a v e b e e n s t u d i e d e x p e r i m e n t a l l y a p p e a r to f o r m v e g e t a t i v e r e p r o d u c t i v e s t r u c t u r e s in r e s p o n s e to (1) c h a n g e s

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in photoperiod, ( 2 ) changes in nutrient availability, or ( 3 ) desiccation ( Sculthorpe, 1967). Knowledge of which environmental factor regulates initiation of vegetative propagule production or senescence for a given species would be useful in management schemes, since it might allow the timely application of control measures, or perhaps point to more effective methods for reducing the quantity of vegetative propagules produced. This in turn might lead to reduced densities of the target plant in the next growing season. Such a scheme (e.g. drawdown) has been proposed for control of Hydrilla verticillata ( L.f. ) Royle in reservoirs in northern Florida (Hailer et al., 1976). Two species of Potomogeton, P. nodosus Poir and P. pectinatus L., grow in irrigation systems in the western U.S.A. and are important weeds in these systems. P. nodosus produces winter buds and P. pectinatus produces tubers at the ends of rhizomes. These structures appear to be the primary means of surviving the winter, when water is usually absent in many irrigation canals (Yeo, 1965 ). Changes in photoperiod are likely to be a more reliable signal of seasonal environmental change than other factors ( Sculthorpe, 1967). Therefore, we examined growth and vegetative propagule production by P. nodosus and P. pectinatus when they were grown at photoperiods of 10, 12, 14 and 16 h, in order to determine if changes in these characteristics were influenced by photoperiod. In addition, we measured the levels of chlorophyll a and b and carotenoids to test a hypothesis suggested by Drew (1979) that leaf senescence may be triggered by changes in photoperiod. MATERIALS AND METHODS

Winter buds of P. nodosus used in this study were originally co!lected from the Richvale Irrigation District California ( 39 ° 30'N latitude, 122 ° 15'W longitude) in February 1985, and stored in the dark at ca. 4°C for 6 weeks before use. P. pectinatus tubers were obtained from Kester's Wild Game Food Nursery, Omro, Wisconsin, ( 48 ° 44' N latitude 44 ° 02' W longitude), and also stored in darkness at 4 ° C until use. Tubers or winter buds were allowed to germinate in 2.8-1 Fernbach flasks, containing well water, for 1 week at 25 ° C in a growth chamber set at a 14:10 (L:D) cycle. Light was provided by cool-white fluorescent lamps at 250/IE m -2 s-1. Following germination, the fresh weight of each sprouted propagule was determined. Fresh weight was 0.412 ___0.169 g for P. nodosus and 0.264_+0.037 g for P. pectinatus, ( m e a n + S . D . ; n = 1 2 8 ) . The sprouted propagules were then planted (one per pot) at 2.5 cm depth in 1-1 plastic pots filled with a mixture of 90% sand and 10% peat (v/v). This mixture was supplemented with KNO3 {0.124 g kg-1), K2SO4 (0.082 g kg-1), dolomite (1.95 g kg-~), gypsum (0.495 g kg -~) and superphosphate (0.879 g kg-1). The pots were placed in 58 × 58 × 33-cm ( inside measurements) plastic tanks filled with well water. The water depth was 28 cm, and the distance from the

105 top of the pot to the water surface was 13 cm. The tanks were placed on tables in a greenhouse under banks of four 2.4-m fluorescent lamps (Sylvania F96T12/GR/VHO) on a 14:10 (L:D) regime. Two tanks containing 16 pots each were used for each photoperiod and each species. The plants were allowed to grow under these conditions for 2 weeks, after which the tables were draped with 1-mm-thick opaque black plastic sheets, so that the photoperiod could be independently regulated for each table, and the photoperiod was set at either 10, 12, 14 or 16 h. The irradiance at the surface of the water was 80 ~E m-2 s - 1 (PAR), as determined with a spherical collector (Biospherical Instruments Model QSL-100). Water in the tanks was replaced every 2-3 days. Daytime water temperature in the tanks, measured daily, averaged 24 °C and did not differ by more than 2-3 oC between photoperiod regimes. Diurnal temperature fluctuations within a tank were also small, 2-3 ° C. At 2-week intervals, 8 pots were removed at random from each photoperiod and the plants were gently washed over screens ( 3-mm mesh) to remove the potting medium and to prevent loss of plant material. Plants were separated into roots and shoots, and the fresh weight determined. Subsamples of plant material were dried at 80 ° C for 24 h, and the ratio of dry weight to fresh weight was used to convert all fresh weights to dry weights. The number ofpropagules per plant and the individual weight of each were measured. Relative growth rates (RGR) for total dry weight were calculated by regressing the natural log of total dry weight vs. time. RGR's have the advantage of allowing comparisons of growth for plants of different size (Hunt, 1982). Within a species, the RGR's at different photoperiods were compared using the General Linear Models procedure in SAS to perform the Test for Heterogeneity of Slopes ( Freund and Littel, 1981; SAS Institute, 1982). Triplicate 150-mg fresh-weight samples of leaves were extracted in 90% acetone for determination of chlorophyll a, chlorophyll b and carotenoids. Absorbance at 665, 645,630 and 480 nm was measured on a Beckman Model 25 spectrophotometer which has a resolution of > 0.2 nm. Pigment concentrations were calculated using the equations provided in Wetzel and Likens (1979) and were expressed per gram fresh weight. The entire experiment was repeated once, but pigment measurements were only made during the second experiment. Pigment levels were analyzed by a two-way analysis of variance, followed by means comparisons using Tukey's HSD procedure ( SAS Institute, 1982). Indication of statistically significant differences are at the 5% level. RESULTS The RGR for P. pectinatus was not significantly affected by photoperiod, but tuber production was enhanced for plants grown at a photoperiod of < 12 h. Mean biomass per plant (Fig. 1 ) increased over time, with RGR's ranging from 16.4+5.4 (slope+S.E.; n = 3 0 ) to 25.5__6.3 (slope+S.E.; n = 2 9 ) mg

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Fig. 1. Growth responses for P. pectinataus. Panel A shows mean plant dry weight (mg), B shows mean number of tubers per plant, C shows mean weight of tubers per plant (mg), and D shows percentage of plants producing tubers. g - 1 d - 1. The mean number of tubers per plant was greater for plants grown at a photoperiod of ~<12 h (Fig. 1). The m a x i m u m mean number of tubers per plant was 12, and was observed for plants grown for 56 days under a 10-h photoperiod. Mean weight of tubers per plant displayed a similar response ( Fig. 1). The proportion of plants grown at a 10-h photoperiod which had tubers increased from 50% at Day 28 to 100% by the 56-day sample. All of the plants grown at a 12-h photoperiod had also produced tubers by Day 56, but ~<25% of those grown at 14- or 16-h photoperiods had produced tubers by Day 56. The response of P. nodosus to photoperiod was similar to that of P. pectinatus, except that R G R differed with photoperiod. Mean dry weight per plant increased after 28 days for plants grown at a 10- or 16-h photoperiod, which had R G R ' s of 19.2_+10.0 ( n = 3 0 ) and 23.1_+6.9 ( n = 3 1 ) m g g - l d a y -], respectively. Plants grown under the 12- and 14-h photoperiods declined, or increased only slightly in dry weight over time, with the result that the R G R was - 4.9 _+7.2 ( n = 31 ) and 0.1 _+ 12.3 ( n = 29), respectively. The mean number of winter buds per plant varied from 5 tO 0.3 for plants grown at a 10- or 12-h photoperiod, respectively. No winter buds were produced by plants grown at 14- or 16-h photoperiods (Fig. 2 ). T h e mean weight of winter buds per plant had a similar response (Fig. 2 ). The proportion of plants producing tubers was 75% in the 28-day sample, and had increased to 100% by Day 56. Only one plant produced winter buds under a 12-h photoperiod. The mean root to shoot ratio for P. pectinatus was 0.10 _+0.04 ( n = 124), and displayed no consistent change with photoperiod. The root to shoot ratio of P.

107

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nodosus was 0.26 + 0.12 (mean + S.D.; n = 126) when averaged over all combinations of time and photoperiod. P. pectinatus tubers accounted for 5% of plant dry weight by Day 28, and 38% by Day 56, for plants grown at a 10-h photoperiod. P. nodosus grown under a 10-h photoperiod had allocated 5% of total dry weight to winter buds by Day 28 and 27% by Day 56 (Fig. 3). Chlorophyll a, carotenoids and the ratio of carotenoids to chlorophyll a were affected by photoperiod and time {sampling day) in a statistically significant way for both P. nodosus and P. pectinatus (Figs. 4 and 5). For P. nodosus, chlorophyll a and carotenoid content declined for plants grown at a 10- or 12h photoperiod, after 27 days exposure. Lack of significant changes in chlorophyll a or carotenoid content for similar plants grown at a 14- or 16-h photoperiod indicated that decreased pigment levels were due to prolonged exposure to short days. The ratio of carotenoids to chlorophyll a generally increased over time, and was greatest for plants grown for 59 days under a 10-h photoperiod. Variations in chlorophyll a:b were small (overall coefficient of variation (C.V.) = 13% ), and were not statistically related to either photoperiod or time. The response of P. pectinatus was somewhat different. Chlorophyll a and carotenoid content for plants grown at a 10-, 12-, or 14-h phot0Period were not significantly different on any sampling date. Plants grown at a 16-h photoperiod displayed a significantly reduced content of both total chlorophyll and carotenoids after 27 days exposure. The ratio of carotenoids to chlorophyll a increased slightly over time, with the greatest change observed for plants grown under a 16-h photoperiod.

108

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Fig. 3. Distribution of dry matter among roots, shoots and vegetative propagules for P. pectinatus (A) andP. nodosus (B).

C o m p a r i s o n o f o v e r a l l m e a n s i n d i c a t e d t h a t c h l o r o p h y l l a for P. pectinatus ( m e a n = 1005 #g g - 1; C.V. = 22%; n - - 35) w a s n e a r l y t w i c e t h a t of P. nodosus ( m e a n = 578/~g g - 1; C.V. = 27 %; n = 34 ). S i m i l a r l y , t h e c a r o t e n o i d c o n t e n t w a s g r e a t e r for P. pectinatus ( m e a n = 238 #g g-1; C.V. = 20%; n = 35 ) t h a n for P. nodosus ( m e a n = 164 #g g - 1; C.V. = 26%; n = 34 ).

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The results presented here demonstrate that tuber or winter-bud production was enhanced by exposure to short days (photoperiod of < 12 h) for both P. pectinatus and P. nodosus. This response is similar to that reported for HydriUa verticiUata (Van et al., 1978; Sutton et al., 1980; Klaine and Ward, 1984, Spencer and Anderson, 1986), Utricularia vulgaris L. (Winston and Gorham, 1979), Myriophyllum verticillatum L. (Weber and Nooden, 1974) and Hydrocharis morsus-ranae L. (Terras, 1900) which have been reported to form vegetative propagules in response to short days. In contrast, P. crispus L. produced turions under long days, i.e. those with photoperiods of >~16 h (Chambers et al., 1985). The root to shoot ratios measured in this study for P. nodosus are similar to those reported by Barko et al. (1982) for plants grown at 20-28 ° C over a range of light intensities. The results of the present study indicate that P. nodosus grown under short days had a greater proportion of total dry weight present as shoots than as roots when compared to plants grown under long day conditions (Fig. 3 ). A comparison of the overall root to shoot ratios for P. nodosus and P. pectinatus indicated that P. pectinatus produced less root tissue per unit of shoot tissue. Winter bud production by P. nodosus was accompanied by a decrease in the production of roots and, to a lesser extent, shoots, whereas the pattern of allocation in P. pectinatus was less clear. For example, winter bud formation in P. nodosus was associated with a decrease of ~ 65% for root biomass, but only c. 15% for shoot biomass. On average, a single P. pectinatus plant produced more

110

vegetative propagules (12 tubers/plant) than P. nodosus (5 winter buds/ plant) for plants grown under similar conditions. However, the weight of an average tuber (13 mg dry weight) was about half that of an average winter bud ( 29 mg dry weight). These data suggest that on a per plant basis, both species invest about the same a m o u n t of photosynthate in perennating structures, but that P. pectinatus apportions this into twice as many, smaller propagules. The potential effect of these apparently different strategies on competitiveness of the two plants must await more detailed examination of propagule longevity, germinability and vigor. In a study of photosynthetic rates of the seagrass Posidonia oceanica, ( L. ) Delite, Drew (1979), noted that leaf chlorophyll content declined markedly in summer, and suggested that leaf senescence (for this species) was probably triggered by changes in photoperiod. The data on pigment composition for P. nodosus support the hypothesis that leaf senescence for some species may be under the influence of photoperiod, as suggested by Drew (1979). A reduction in chlorophyll production under short photoperiods has been reported for several species of terrestrial plants as well (Vince-Prue, 1975). The changes in the ratio of carotenoids to chlorophyll a and the lack of change in chlorophyll a:b further suggest that chlorophyll levels may be more strongly influenced by photoperiod than by carotenoid levels. P. pectinatus pigment composition, however, did not display similar changes. In fact, the measured pigment levels for P. pectinatus grown at a 10-, 12-, or 14-h photoperiod were not statistically different. Pigment levels declined only for plants grown under a 16-h photoperiod. Total chlorophyll values for P. nodosus were less than those reported by Barko and Filbin (1983). However, pigments in this study were extracted with 90% (v/v) aqueous acetone, whereas Barko and Filbin (1983) used dimethyl sulfoxide, which may be more efficient for extracting chlorophyll ( Shoaf and Lium, 1976; Hains, 1985 ). Taken as a whole, the responses to photoperiod measured in this study indicate that even though both species are influenced by photoperiod, P. nodosus appears to be more strongly influenced than P. pectinatus. This conclusion stems from the fact that production of tubers by P. pectinatus occurred, albeit at low levels, under 14- and 16-h photoperiods, and changes in pigment composition were not evident for P. pectinatus grown under short days. The plants in this study were grown from propagules that were originally collected from quite separate locations, thus, care must be taken when comparing their responses to photoperiod. It is somewhat unexpected, however, that P. pectinatus from a more northern location appeared to be less strongly influenced by photoperiod than did P. nodosus which was from the more southern location. To our knowledge, studies indicating the existence of photoperiod-related ecotypes within a species, such as those demonstrated for flowering in some terrestrial plants (Raven et al., 1981 ), do not exist for aquatic macrophytes. Unfortunately, the present study does not address this question either,

111

so it is not possible to compare the differences between P. nodosus and P. pectinatus with what might be expected for populations of either species along a latitudinal gradient. It is noteworthy, however, that plants of the monoecious strain of H. verticillata, which to date has been found in more northern locations in the U.S.A. than the dioecious strain, appear to produce subterranean turions (tubers) more rapidly than the dioecious plants when both are grown under short photoperiods. (Spencer and Anderson, 1986). ACKNOWLEDGMENTS

We thank Drs. J. McHenry and J. Titus and two anonymous reviewers for commenting on an earlier version of this manuscript. We also thank Ms. D. Gee, Mr. R. Sedlacek, Mr. J. Shaff, Ms. C. Cowan and Ms. S. Fellows for help in processing the plants. Dr. M. Ames and Mr. N. Dechoretz also provided valuable assistance.

REFERENCES Barko, J.W. and Filbin, G.J., 1983. Influences of light and temperature on chlorophyll composition in submersed freshwater macrophytes. Aquat. Bot., 15: 249-255. Barko, J.W., Hardin, D.G. and Mathews, M.S., 1982. Growth and morphology of submersed freshwater macrophytes in relation to light and temperature. Can. J. Bot., 60: 877-887. Chambers, P.A., Spence, D.H.N. and Weeks, D.C., 1985. Photo-control of turion formation by Potamogeton crispus L. in the laboratory and natural water. New Phytol., 99: 183-194. Drew, E.A., 1979. Physiological aspects of primary production in seagrasses. Aquat. Bot., 7: 139-150. Freund, R.J. and Littel, R.C., 1981. SAS for Linear Models. SAS Institute, Carey, NC, 231 pp. Hains, J.J., 1985. Practical considerations for routine chlorophyll measurements: precautions and comparison of extraction methods. J. Freshwater Ecol., 3: 175-180. Hailer, W.T., Miller, J.L. and Garrard, L.A., 1976. Seasonal production and germination of hydrilla vegetative propagules. J. Aquat. Plant Manage., 14: 26-29. Hunt, R., 1982. Plant Growth Curves: The Functional Approach to Plant Growth Analysis. University Park Press, Baltimore, MO, 248 pp. Hutchinson, G.E., 1975. A Treatise on Limnoiogy. Vol. III, Limnological Botany. John Wiley, New York, 660 pp. Klaine, S.J. and Ward, C.H., 1984. Environmental and chemical control of vegetative dormant bud production in HydriUa verticiUata. Ann. Bot., 53: 503-514. Raven, P.H., Evert, R.F. and Curtis, H., 1981. Biology of Plants (3rd edn). Worth, New York, 686 pp. SAS Institute, 1982. SAS User's Guide: Statistics. SAS Institute, Cary, NC, 923 pp. Sculthorpe, C.D., 1967. The Biology of Aquatic Vascular Plants. Arnold, London, 610 pp. Shoaf, T.W. and Lium, B.W., 1976. Improved extraction of chlorophyll a and b from algae using dimethyl sulfoxide. Limnol. Oceanogr., 21: 926-928. Spencer, D.F. and Anderson, L.W.J., 1986. Photoperiod responses in monoecious and dioeciou., Hydrilla verticillata. Weed Sci., 34: 551-557. Sutton, D.L., Littel, R.C. and Langeland, K.A., 1980. Intraspecific competition of HydriUa ver. ticillata. Weed Sci., 28: 425-428.

112 Terras, J.A., 1900. Notes on the germination of the winter buds of Hydrocharis morsus-ranae. Trans. Proc. Bot. Soc. Edinb., 21: 318-329. Van, T.K., Haller, W.T. and Garrard, L.W., 1978. The effect of day length and temperature on Hydrilla growth and production. J. Aquat. Plant Manage., 16: 57-59. Vince-Prue, D., 1975. Photoperiodism in Plants. McGraw-Hill, Maidenhead, 444 pp. Weber, J.A. and Nooden, L.D., 1974. Turion formation and germination in Myriophyllum verticillatum: Phenology and its interpretation. Mich. Bot., 13:151-158. Wetzel, R.G. and Likens, G.E., 1979. Limnological Analysis. Saunders, Philadelphia, PA, 357 pp. Winston, R.E. and Gorham, P.R., 1979. Turions and dormancy states in Utricularia vulgaris. Can. J. Bot., 57: 2740-2749. Yeo, R.R., 1965. Life history of sago pondweed. Weeds, 13: 314-321.