Environmental factors affecting seed germination in Myriophyllum spicatum L.

Aquatic Botany 45 (1993) 15-25 Elsevier Science Publishers B.V., Amsterdam

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Environmental factors affecting seed germination in Myriophyllum spicatum L. Christopher F. Hartleb l, John D. Madsen 2 and Charles W. Boylen Department of Biology and Fresh Water Institute Rensselaer Polytechnic Institute Troy. NY 12180, USA (Accepted 28 October 1992 )

ABSTRACT Hartleb, C.F., Madsen, J.D. and Boylen, C.W., 1993. Environmental factors affecting seed germinalion in Myriophyllum spicatum L. Aquat. Bot., 45:15-25.

Myriophyllum spicatum L. is a submersed aquatic macrophyte that has colonized aquatic habitats across North America. Vegetative propagation is prevalent in M. spicatum and is highly visible, because of the extraordinary capacity of this plant to spread by fragmentation. Because of this. sexual propagation has been considered of limited importance and little is known about in situ seed germination of this species. Laboratory studies were conducted on seeds of M. spicatum to determine the light and temperature requirements necessary for maximum seed germination. Seed germination was also examined in situ at depths of 1, 3 and 5 m in two bays at Lake George, New York (USA). Significant germination of seeds was observed in the northern bay of the lake, but not in the southern, probably because of increased sedimentation in the latter. Temperatures higher than 1 5 C were found to be necessary for successful seed germination in laboratory studies; however, light was determined not to be a limiting factor on its own. Seeds buried under more than 2 cm of sediment were shown to experience significant decreases in germinalion.

INTRODUCTION

Flowering ofMyriophyllum spicatum L. occurs once or twice annually, usually in mid to late July and in August (Kimbel, 1982). Flowers are tetramerous, with the fruits splitting into nutlets containing only one seed. A young population of M. spicatum in Lake George produced a median of seven whorls of female flowers, or 28 total flowers, for a total possible seed set of 112 seeds per stalk (Madsen and Boylen, 1988). The percentage of seed set may vary Correspondence to: C.W. Boylen, Department of Biology and Fresh Water Institute, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. ~Present address: Department of Zoology, University of New Hampshire, Durham, NH 03824. USA. 2Present address: US Army Engineer Waterways Experiment Station, Lewisville Aquatic Ecosystem Research Facility, R R No. 3 Box 446, Lewisville, TX 75056, USA.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-3770/93/$06.00

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C.F. HARTLEB ET AL.

tremendously both between lake sites and between years (J.D. Madsen, unpublished data, 1990). Despite the high seed production, no seedlings have been observed in nature, not has any seed germination been documented in situ. Vegetative propagation is prevalent in M. spicatum and is highly visible as a result of the intense capacity of this plant to spread by vegetative fragmentation. Because of this, sexual reproduction has been considered of limited importance (Aiken et al., 1979). As a result, little is known about the sexual reproduction of this species and of the potential that seed germination represents as a colonization factor. IfM. spicatum is able to create a large seed bank that survives long periods of dormancy and disturbance, then viable germinating seeds would reduce the effectiveness of weed management activities such as herbicide treatments, lake drawdown and dredging (McDougall, 1983). Local variation in the density of ecologically active seeds can be related to many environmental gradients such as depth, sediment texture, irradiance, temperature and disturbance (Patten, 1956; Kimbel, 1982; Haag, 1983 ). The effects of depth and sediment texture on species composition may be as important as their effect on the ecophysiology of the species in determining the size and composition of the seed bank at a particular site (Haag, 1983). This study was designed to examine the environmental gradients that affect seed germination ofM. spicatum. Seeds were collected from dense beds of M. spicatum and transferred to controlled study areas in situ, at depths of 1, 3 and 5 m and observed for a 1-month period. At the end of that month the seeds and seedlings were collected and the per cent germination of seeds was determined. Seeds were also transferred to the laboratory, where they were germinated under controlled temperature and photoperiods. Poor seed germination in the presence of high deposition prompted further laboratory studies to investigate the effect of sedimentation on germination. METHODS

Study site description Lake George is located in northeastern New York State, USA (Fig. 1 ). It is a large ( 114 km 2), deep ( 58 m maximum depth ) and relatively clear (Secchi disk depth 9.2 m) oligotrophic lake, with a circum-neutral pH (7.58) and low alkalinity (15-20 mg CaCO3 l - t ) (Madsen et al., 1988). The M. spicatum population in Lake George is fairly young; significant populations were first observed in 1985 (Madsen and Boylen, 1989). Huddle Bay, a large shallow embayment with heavy shoreline development, and Northwest Bay, a large intermediate depth embayment with minimally developed shoreline, were used as in situ study sites (Fig. 1 ).

SEED GERMINATION OF MYRIOPHYLLUM SPICA TUM L.

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Lake George

New York State Northwest Bay Brook

Huddle Bay

8 km Lake George

Fig. 1. Map of Lake George (NY, USA), showing the location ofMyriophyllum spicatum L. in situ study sites.

Seed collection and sterilization Flowering stalks for laboratory seed experiments along with approximately 20 cm of vegetative stem material were collected in August 1988. The stalks were incubated in aquaria with Lake George water under controlled light (400 /tmol m -2 s - l photosynthetically active radiation (PAR), 14 h light: l0 h dark) and temperature (20 _+2 °C). Flowering stalks were maintained until seed set and maturity were achieved, a period of approximately 4 weeks. Pollination was assumed to have occurred before collection. Seeds were collected from emergent fruiting spikes, quantified and allowed to overwinter at 4 °C for 4 months. The in situ cold period for Lake George is approximately 5 months. O p t i m u m sterilization time for laboratory germination experiments was previously determined (Hartleb, 1990). Five groups of 100 seeds, representing sterilization times of 0, 5, 15, 25 and 35 rain, were surface sterilized in 5.25% sodium hypochlorite, rinsed with sterilized distilled water and placed in sterile Petri dishes. The dishes were placed under a 14 h light: 10 h dark daylength cycle, with a light intensity of 200/tmol m -2 s - l PAR. Temperature was maintained at 20°C in a controlled growth room. The number of germinating seeds in each dish was counted daily for a period of 2 weeks. O p t i m u m sterilization time was also dependent on the time necessary to minimize the survival of microbial contaminants. Five groups of ten seeds sterilized as above were placed in Petri dishes containing nutrient agar. The dishes were placed in an incubator, at 35 °C, for a period of 3 days, after which

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C.F. HARTLEB ET AL.

they were examined for the growth of any bacteria, algae and fungi. The absence of contaminant growth was used to establish the o p t i m u m sterilization time for the laboratory seed germination experiments. Maximum seed germination was observed after 7 days for all treatments in 5.25% sodium hypochlorite. Germination was highest for seeds treated for 25 min with sodium hypochlorite. An examination of contamination by bacteria, algae and fungi showed minimal contamination at the 25 min period.

Laboratory seed germination A total of 1500 seeds were surface sterilized for 25 min in 5.25% sodium hypochlorite as established before. Temperature and daylength treatments were performed on groups of 500 seeds, which were germinated in Petri dishes containing sterilized distilled water. Three daylength periods were examined ( 10 h light: 14 h dark; 14 h light: 10 h dark; 0 h light:24 h dark) in combination with five temperature regimes (5, 10, 15, 20 and 25 °C). Five plates containing 20 seeds each were placed at each temperature treatment and five groups of five plates were placed at each daylength to give a total of 500 seeds at each daylength treatment. The dishes were placed in growth chambers to maintain constant temperatures and the light intensity was set at 200/tmol m - 2 sPAR. At the end of the experimental period (established at 1 week during sterilization experiments), the seeds were removed and analyzed for germination, which was indicated by the emergence of a root tip from a cracked seed coat.

In situ seed germination In situ germination containers were prepared using plastic cups, which were filled with washed fine sandy sediment, m o u n t e d in trays (weighted to keep the cups on the lake bottom), and covered with fine-mesh screening to contain the diminutive seeds ( 300 mg dry weight (DW), 2 m m length; J.D. Madsen, unpublished data, 1990). A marker buoy was attached to each tray, so that the trays could be located and recovered. Ten seeds were placed in each cup ( 10 cups per tray, two trays per depth), covered with screening and placed at depths of 1, 3 and 5 m outside a milfoil bed in both Huddle and Northwest Bays on 9 June 1989. After a 1-month germination period, the trays were collected (11 July 1989) and the number of germinated seeds was counted. Per cent germination was calculated for seeds from each depth and from each embayment.

Effect of burial on germination The effect of burial by sedimentation on the germination of seeds was examined. Experiments were performed during the first 2 weeks of August 1989.

SEED GERMINATION OF MYRIOPtlYLLUM SPI('I7+UM L.

[9

Containers, of height 15 cm, were filled with sandy sediment and placed in aquaria in the laboratory. A strip of dialysis tubing, of length 17 cm, was divided into l-cm sections, and into each section five M. spicatum seeds were placed. A total of six strips were prepared and placed in the containers, with 13 cm of the dialysis tubing being covered with sediment and 3 cm being above the surface, and one section at the sediment-water interface. Dialysis tubing was used as an inert seed holder that would confine seeds to maintain their position and allow easy retrieval, yet not restrict the flow of water or nutrients. Temperature was maintained at 20°C and light intensity at 400 /lmol m -2 s-1 PAR, with daylength at 14 h light: 10 h dark. After a 1-week germination period the tubing was removed and the number of germinated seeds in each section determined. Tetrazolium testing for seed viability had been attempted in earlier work as a means of evaluating seed stock viability and reduction in germination because of dormancy. However, repeated trials failed to produce a replicable method (Madsen and Boylen, 1988 ). RESULTS

Laboratory seed germination Maximum seed germination was observed at a temperature of 20 °C and daylength of 14 h light: l0 h dark (Fig. 2). Almost no germination was observed for all daylengths at the temperature regimes of 5 and 10°C. Z 2 tests for seed germination indicated that there was a highly significant difference in germination at all temperatures exposed to the daylength of 14 h light: 10 h dark ( P < 0.0001 ). The effect of daylength at 10 h light:14 h dark was also highly significant across all temperatures based on Z 2 tests ( P < 0.0001 ). The

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Fig. 2. Percentageof seeds germinatedduring laboratory experimentsto determineoptimum temperatureand daylength.Bars with differentletters are significantlydifferentat the P=0.05 level, as determinedby two-by-twoZ2tests.

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C.F. HARTLEB ET A L

effect of absence of light (dark treatment) on seed germination across all temperature regimes was highly significant based on Z 2 tests ( P < 0.0001 ). Seed germination in the presence of light was greater for the three higher temperatures ( 15, 20 and 25 ° C), than germination in the dark treatment within each temperature regime. The highest per cent germination was observed in the 20 °C temperature treatment for all daylength periods.

In situ seed germination Per cent seed germination in Northwest Bay was significantly greater than seed germination in Huddle Bay (z2P< 0.0001; Fig. 3 ). Seed germination at all three depths ( l, 3 and 5 m) in Northwest Bay was significantly higher than seed germination in Huddle Bay at all three depths based on Z2 tests ( P < 0.0001 ). In both bays the highest rate of seed germination was observed at the 5 m depth, with no significant difference in germination between the 1 and 3 m depths. 5O DEPTH

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Fig. 3. Percentage of seeds germinated for each bay study site at depths of l, 3 and 5 m. Bars with different letters are significantly different at the P = 0 . 0 5 level, as determined by two-bytwo g 2 tests. -I-/I////////I///////I ~7 Lfl --

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Fig. 4. Percentage of seeds germinated vs. the depth of burial of the seeds in sediment. Bars with different letters are significantly different at the P = 0.05 level, as determined by two-by-two g 2 tests.

SEED GERMINATION OF MYRIOPHYLLUM SPIC4TUM L.

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Effect of burial on germination Per cent germination of seeds was significantly greater for seeds buried under less than 2 cm of sediment, or for seeds not buried at all, based on Z 2 tests ( P < 0.001, Fig. 4). Germination was significantly reduced, to less than 15%, once the depth of seed burial exceeded 2 cm. Seeds placed above the sediment surface showed the greatest per cent germination, with up to 60% germinating. DISCUSSION

Seeds which were exposed to lower-temperature treatments (5 and 10 °C) exhibited lowest rates of germination across all daylength exposures. Myriophyllum spicatum seeds require a temperature higher than 10 °C before they begin to show significant germination rates. The requirement for water temperature higher than 10 ° C may have adaptive significance. Potamogeton pectinatus L. tubers require temperatures of at least 15 ° C to germinate (Madsen and Adams, 1988a), which corresponds to the temperature at which it demonstrates positive carbon balance (Madsen and Adams, 1988b). Also, laboratory photosynthesis studies have shown that daily carbon balance ofM. spicatum is lower at 15°C than at 20°C (Madsen and Boylen, 1990). Seeds exposed to the higher temperatures of 15, 20 and 25 °C and exposed to dark treatments showed an average of 40% germination. It has been shown before that some seeds of M. spicatum will germinate in the dark, regardless of light treatment, given the proper temperature and pretreatment conditions (Madsen and Boylen, 1988). Therefore, the absence of light alone does not prevent seed germination, and a combination of another factor (s) plus light limitation must be responsible for the lack of seed germination in nature. Both 14 h light: 10 h dark and 10 h light: 14 h dark daylength treatments showed significant seed germination in the higher-temperature regimes of 15, 20 and 25°C, with per cent germination around 50% (Fig. 2). Maximum germination was achieved at 20°C and 14 h light:10 h dark, and this corresponds to the temperature and daylength experienced at Lake George during the late spring and early summer months. Therefore, it would be expected that seed germination should have occurred in Lake George, unless other limiting factors were present to prevent germination. A comparison of per cent seed germination in Northwest Bay and Huddle Bay (Fig. 3 ) illustrates the requirements for suitable conditions to be present for seeds to germinate. Huddle Bay, which has a heavy shoreline development, experiences much use and recreational activity, and is affected by nutrient runoff from a nearby brook and marsh (Eichler and Boylen, 1988). There is a constant churning of the water, and sediment particles are in continuous motion during most of the day. During the course of the experiment over 3 cm of sediment was deposited on top of the containers and seeds. My-

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C.F. H A R T L E B ET AL.

riophyllum spicatum seeds were shown to exhibit significant germination if covered with 2 cm of sediment or less. It is likely that buried seeds remain ungerminated if covered in sediment. Upon disturbance of the sediment and exposure to illuminated conditions, dormant seeds may be induced to germinate and colonize the disturbed site, as occurs in Halophila engelmanii Aschers. (McMillan, 1987). Submersed aquatic plants reflect a particularly strong degree of dormancy and may remain dormant for several years until suitable conditions prevail (Haag, 1983 ). Seeds in Northwest Bay exhibited far higher germination rates (over 30%) than seeds from Huddle Bay (only 5%). Northwest Bay, with its less developed shoreline, experiences far less recreational activity and there is less sedimentation from streams and marshes (Eichler and Boylen, 1988 ) than at Huddle Bay. Seeds deposited in water not subject to the effects of severe wavewashing and sedimentation may experience significant germination rates (McDougall, 1983 ). Comparison of germination rates by water depth in Northwest Bay and Huddle Bay indicates that seeds prefer the 5 m depth for germination in both bays (Fig. 3 ). In Northwest Bay there is a 10% difference in germination rates between the 5-m site and the 1-m and 3-m sites. In Huddle Bay the 5 m site experienced the greatest germination rate, followed by the 3 m and then the 1 m sites. This trend, of deeper sites showing the greatest germination rates, once again reflects the need by the seeds for calm, undisturbed areas in which to germinate. The 5 m depth provided the undisturbed conditions the seeds required to germinate, whereas the l-m and 3-m sites had too much wave and recreational action for the seeds to experience significant germination rates. However, M. spicatum seeds germinating at a depth of 5 m will probably not form dense beds. Natural progression in the colonization of milfoil within a lake dictates that after scattered plants have established themselves new plants will increase the density within the established area (Madsen et al., 1991 ). Myriophyllum spicatum has been shown to grow at depths between 1 and 4 m, with optimal growth at 3 m (Madsen et al., 1989), so even though maximum germination occurred at the 5-m depth colonization by seedlings is doubtful. These results, when combined with the laboratory results that showed significant seed germination in dark treatments at high temperatures, tend to disagree with previous findings that light qualities and quantities strongly influence the germination rate of milfoil seeds (Coble and Vance, 1987 ). Our results, as well as those by Madsen and Boylen ( 1988 ), show that absence of light is not the only factor limiting the germination of seeds. Viable seeds may have multiple chilling requirements, or be buried too deeply for germination to occur. Cues such as sediment texture (nutrient content) or the appropriate redox potential may be lacking, or some process such as breakdown of fruits by bacterial action or gut passage through birds may need to occur before seeds will germinate (Haag, 1983 ).

SEED GERMINATION OF MYRIOPHYLLUM SPICAFUll L.

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Experiments on seed germination after burial in sediment showed that seeds buried deeper than 2 cm below the surface exhibited a significant decrease in germination (Fig. 4). Seeds that were placed above the sediment surface showed germination rates greater than 50%, whereas those seeds located between the sediment surface and sediment depth of 2 cm showed germination rates greater than 30%. Once the seeds were placed below 2 cm of sediment, germination rates dropped below 10%. Therefore, sedimentation is a key factor limiting the germination ofM. spicatum seeds and, as shown above, seeds located in Huddle Bay, which had the highest rates of sedimentation, experienced the lowest rates of germination. Many factors may be limiting the germination of these buried seeds, such as the lack of sufficient light, mechanical action, oxygen tension, absence of bacterial action on seeds, or the decrease in temperature experienced at greater depths in sediment. The laboratory experiment was carried out in homogeneous sediment of fine sand and under controlled temperature conditions, so some of these factors were eliminated, yet significant germination still did not occur below 2 cm of sediment. The maximum germination of seeds in Lake George of only 40% may indicate that seed production and germination might not appear to be sufficient to contribute to the spread of the population in this lake. There are lakes in which M. spicatum exhibits higher seed set percentages (Madsen and Boylen, 1989), and seed germination may contribute to the annual propagation and expansion of the populations in these lakes. Seeds may remain dormant for prolonged periods of time and germinate only when favorable conditions prevail, such as suitable depth, water temperature, adequate substrate, water quality and protection from excessive wave exposure (Collins et al., 1987). Therefore, M. spicatum seeds may represent a long-term survival adaptation for this species, or a case of"bet-hedging" (sensu Crawley, 1986 ) in a reproductive strategy dominated by vegetative propagation and perennation. CONCLUSIONS

Seed germination ofM. spicatum does indeed occur in situ, but is affected by many factors. Laboratory studies showed that temperature and daylength are both factors that affect the percentage of seeds that will germinate, but dark conditions alone will not prevent seeds from germinating. Sedimentation causes seeds to show a significant decrease in germination, probably because it induces many other limiting factors upon the successful germination of the seeds; however, this is an area that needs further study to determine its overall impact on the inhibition of germination. The seed of M. spicatum is well endowed with a remarkable survival capacity mediated through its germination habit and adaptations which insure that seedlings are produced only when favorable conditions for maturation prevail and that rapid germination

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will proceed concurrent with the inception of such conditions (Patten, 1955 ). The seed of M. spicatum does not tend to germinate soon after its development unless exposed to certain conditioning treatments. Under natural conditions, it is likely that low winter temperatures and occasional freezing inhibit germination, and at the same time increase after-ripening, so that prompt germination is insured with spring warming (Patten, 1955 ). The survival of M. spicatum seedlings in situ is probably restricted by factors such as grazing, competition and shading from other aquatic plants, water depth and exposure to wave action. Under suitable conditions, M. spicatum seeds can germinate and grow to mature size (McDougall, 1983 ). There are several plausible explanations that may account for the low numbers or lack of seedlings found in nature. Many seedlings may have been overlooked as it is difficult to identify them among the extensive and intertwined growth of germinating turions, rhizomes and root crowns of other plants, as well as within an M. spicatum bed itself. Also, seedlings may not be capable of competing with plants from turions which germinate and grow rapidly and in profuse numbers early in spring (Scribailo and Posluszny, 1985 ). In general, M. spicatum has a strong potential to develop a large seed bank, and seeds could provide genetic diversity and may represent a long-term adaptation to species survival. Sexual reproduction by seeds provides a means of reestablishment after disturbance and may lead to local changes in density during years favorable for seed production and germination. ACKNOWLEDGMENTS

Primary funding was provided by a Darrin Summer Scholarship to Christopher F. Hartleb. Additional support was provided by the United Parcel Foundation. This paper is contribution 565 of the Rensselaer Fresh Water Institute.

REFERENCES Aiken, S.G., Newroth, P.R. and Wile, I., 1979. The biology of Canadian weeds 34. Myriophyllum spicatum. L. Can. J. Plant Sci., 59:201-215. Coble, T.A. and Vance, B.D., 1987. Seed germination in Myriophyllum spicatum L. J. Aquat. Plant Manage., 25: 8-10. Collins, C.D., Sheldon, R.B. and Boylen, C.W., 1987. Littoral zone macrophyte community structure: distribution and association of species along physical gradients in Lake George, New York, U.S.A. Aquat. Bot., 29: 177-194. Crawley, M.J., 1986. Life history and environment. In: M.J. Crawley (Editor), Plant Ecology. Blackwell Scientific, London, pp. 253-290. Eichler, L.W. and Boylen, C.W., 1988. Final report on the Lake George inshore chemical monitoring program. Rensselaer Fresh Water Institute Rep. 88-3. Rensselaer Polytechnic Institute, Troy, NY, 16 pp.

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Haag, R.W., 1983. Emergence of seedlings of aquatic macrophytes from lake sediments. Can. J. Bot., 61: 148-156. Hartleb, C.F., 1990. Physiological ecology of seed germination in Myriophyllum spicatum L. Rensselaer Fresh Water Institute Rep. 90-15. Rensselaer Polytechnic Institute, Troy, NY. 41 pp. Kimbel, J.C., 1982. Factors influencing potential intralake colonization by Myriophyllum spicatum L. Aquat. Bot., 14: 295-307. McDougall, 1.A., 1983. A study of the germination potential in Myriophyllum spicatum L. seeds. Studies on Aquatic Macrophytes Part XXIV. Province of British Columbia Ministry of Environment, Water Management Branch, Victoria, 31 pp. McMillan, C., 1987. Seed germination and seedling morphology of the seagrass, Halophila engelmannii (Hydrocharitaceae). Aquat. Bot., 28:179-188. Madsen, J.D. and Adams, M.S., 1988a. The germination ofPotamogeton pectinatus tubers: environmental control by temperature and light. Can. J. Bot., 66: 2523-2526. Madsen, J.D. and Adams, M.S., 1988b. The seasonal biomass and productivity of the submerged macrophytes in a polluted Wisconsin stream. Freshwater Biol., 20:41-50. Madsen, J.D. and Boylen, C.W., 1988. Seed ecology of Eurasian watermilfoil (Myriophyllum spicatum L. ). Rensselaer Fresh Water Institute Rep. 88-7. Rensselaer Polytechnic Institute, Troy, NY, 25 pp. Madsen, J.D. and Boylen, C.W., 1989. Eurasian watermilfoil seed ecology from an oligotrophic and eutrophic lake. J. Aquat. Plant Manage., 27:119-121. Madsen, J.D. and Boylen, C.W., 1990. The physiological ecology of Eurasian watermilfoil (,~(vriophyllum spicatum L.) and native macrophytes in Lake George: depth distribution ofbiomass and photosynthesis. Rensselaer Fresh Water Institute Rep. 89-6. Rensselaer Polytechnic Institute, Troy, NY, 52 pp. Madsen, J.D., Eichler, L.W. and Boylen, C.W., 1988. Vegetative spread of Eurasian watermilfoil in Lake George, New York. J. Aquat. Plant Manage., 26: 47-50. Madsen, J.D., Eichler, L.W. Boylen, C.W., Sutherland, J.W., Bloomfield, J.A. and Roy, K.M., 1989. The Lake George Aquatic Plant Survey Final Report. NYS Dept. of Environ. Conserv., Albany, NY, 219 pp. Madsen, J.D., Sutherland, J.W., Bloomfield, J.A., Eichler, L.W. and Boylen, C.W., 1991. The decline of native vegetation under dense Eurasian watermilfoil canopies. J. Aquat. Plant Manage., 29: 94-99. Patten, Jr., B.C., 1955. Germination of the seed ofMyriophyllum spicatum L. Bull. Torrey Bot. Club, 82: 50-56. Patten, Jr., B.C., 1956. Notes on the biology ofMyriophyllum spicatum L. in a New Jersey lake. Bull. Torrey Bot. Club, 83: 5-18. Scribailo, R.W. and Posluszny, U., 1985. The reproductive biology ofHydrocharis morsus-ranae. II. Seed and seedling morphology. Can. J. Bot., 63: 492-496.