2008 NORTHEASTERN NATURALIST 15(2):177–188 Natural History of Heterophylly in Nymphaea odorata ssp. tuberosa (Nymphaeaceae) Philip J. Villani1,* and Shelley A. Etnier1 Abstract - Nymphaea odorata (American white water lily) is an aquatic plant that displays pronounced heterophylly, the appearance of different leaf forms on a single plant. Water lilies produce leaves that either float or are held above the water’s surface. In this paper, we describe the natural history of water lily leaf forms and examine some of the factors that stimulate heterophylly. Over the course of a growing season, the predominant leaf form switches from surface leaves in the early season to aerial leaves in the midseason and then back to surface leaves at season’s end. While many factors are known to contribute to heterophylly, our results suggest that changes in the light environment may be the controlling factor in this system. Introduction Heterophylly, the appearance of different leaf forms on a single plant, is a common feature of many plants (Arber 1920, Schlichting 1986, Sculthorpe 1967). Some of the most dramatic examples of heterophylly occur in the aquatic amphibious plants. In these plants, heterophylly occurs when a single plant is growing in two physically dissimilar environments (i.e., aquatic and terrestrial) that pose substantially different metabolic and mechanical demands on separate parts of the organism. Leaf adaptations often reflect the physical differences in these environments and typically include changes in leaf morphology, anatomy, and/or the position of the lamina relative to the water’s surface (for a general review see Sculthorpe 1967). One example of a plant that changes the position of its leaves relative to the water’s surface is Nymphaea odorata Ait ssp. tuberosa (Paine) Wiersma & Hellquist (American white water lily). It is commonly found in still or slow-moving waters in the northeastern parts of North America (Gleason and Cronquist 1991). During growth of the white water lily, its shoot system (i.e., a rhizome) remains buried in the sediment below the water’s surface throughout the life of the plant. Immature leaves are produced from the rhizome and mature into three leaf types: immersed, which remain submerged; surface, which float on the water’s surface; and aerial, which are held above the water (Sculthorpe 1967; Fig. 1). These three leaf forms do not represent a growth continuum, as a developing leaf matures into one form or the other. Immersed leaves occur early in the season (Sculthorpe 1967) and are never abundant (P.J. Villani, pers. observ.). In some populations, the surface leaf is the predominant leaf form, while other populations produce both surface and aerial leaf forms (S.A. Etnier, pers. observ.). 1Department of Biological Sciences, Butler University, Indianapolis, IN 46208. *Corresponding author - pvillani@butler.edu. 178 Northeastern Naturalist Vol. 15, No. 2 In a previous study, we characterized the differences in biomechanical properties between surface and aerial petioles (Etnier and Villani 2007). We found that an aerial leaf rises above the water’s surface due to increased rigidity of its petiole. The increased rigidity is due to subtle changes in petiole anatomy and morphology. There seem to be seasonal differences in the abundance of these two leaf types (S.A. Etnier, pers. observ.), but little is known about the causal factors determining their appearance. Many heterophyllous aquatic species switch between a combination of immersed, surface (floating), and aerial leaf forms, although the exact mechanism for this switch varies (Minorsky 2003). In Nuphar lutea (L.) Sm. (yellow cow lily), herbivory causes an increase in immersed leaf production relative to aerial leaves (Kouki 1993). In other aquatic species, the switch to aerial leaf forms was stimulated by changes in water depth (Horn 1988, Nohara and Kimura 1997), composition of sediment type (Barko and Smart 1986), and changes in the concentration of dissolved carbon dioxide as the shoots grow out of the aquatic into a terrestrial environment (Bristow and Looi 1968, Titus and Sullivan 2001). The exogenous application of abscisic acid, a well-known plant hormone produced in response to osmotic stress, has also been shown to mediate the switch from immersed to aerial leaf forms (Anderson 1978, Liu 1984, Ram and Rao 1982). In other species, features of the light environment including fluency rates (Goliber 1989), light quality (Bodkin et al. 1980, Lin and Yang 1999), and photoperiod (Cook 1969, Kane and Albert 1987, Schmidt and Millington 1968) influenced heterophylly. To our knowledge, no studies have examined the factors influencing heterophylly in the white water lily, although Sculthorpe (1967) briefly mentioned that crowding may induce the aerial leaf form. Figure 1. Heterophylly in the American white water lily as shown in a leaf-removal experiment showing the two different forms of the lily pad leaves: surface (A, center of figure) and aerial (B). Figure 1A is an experimental plot in which leaf removal occurred. Leaves were selectively removed from the experimental plots in order to maintain a 50%-exposed water surface, and Figure 1B is a control plot in which there was no leaf removal. 2008 P.J. Villani and S.A. Etnier 179 In this paper, we investigate the natural history of heterophylly in the white water lily. First, we describe the seasonal distribution of surface and aerial leaves throughout the growing season. Second, we examine some of the factors that may be responsible for the stimulation of heterophylly under natural conditions. Based on research on other heterophyllic plants, we examine differences in life-history traits, including growth rates and leaf longevity. We also examine physical and chemical aspects of the pond, as well as the influence of crowding, on the appearance of surface and aerial leaves. Materials and Methods Plant material All measurements were taken on a population of American white water lily growing in a half-acre ice-skating pond at Eagle Creek City Park, Indianapolis, IN. Observations and experiments were completed during the 2005 growing season, with the exception of the longevity and crowding studies, which were conducted during 2006. The lily pad growing season started at the beginning of April, when leaves first appeared in the pond, and continued until their disappearance from the pond in late October and early November. In this study, we have divided the growing season into three parts: early season was defined as April and May, mid-season as June and July, and late season as August through October. Water chemistry During the growing season of 2006, we measured water chemistry parameters biweekly. Using a LaMotte aquaculture test kit (Chestertown, MD), we determined water pH and dissolved oxygen (dO2), carbon dioxide (dCO2), and ammonia concentrations. Life-history characteristics At the onset of the growing season (early April), six square plots (1 m2) were demarcated within the pond using garden stakes. Three plots were placed on both the east and west side of the pond at 5, 10, and 20 m distances from the shore along a straight line. We counted the number of immature, surface, and aerial leaf forms occurring within the plots two to three times a week throughout the growing season. We also recorded water temperature and depth. We measured the growth rate during the period in which the lamina extends rapidly up to the water’s surface, in both surface and aerial petioles. In each of the plots described above, we selected a small immature leaf close to the pond bottom, marked it by placing a colored twist-tie loosely around its base, and placed a garden stake near the leaf to allow us to relocate it easily. Two to three times a week, we measured petiole length and diameter at the midpoint, lamina length and width, and also recorded observations on the shape of the elongating lamina (e.g., tightly coiled, 180 Northeastern Naturalist Vol. 15, No. 2 loosely coiled, partially open, or fully open). Each marked leaf was monitored until it reached the water’s surface, at which time we discontinued growth measurements. We continued to observe the leaf until its final form (i.e., surface or aerial) could be determined. As soon as we stopped taking measurements on one leaf, we selected another immature leaf and repeated the process. We monitored 75 leaves between April and October. Growth rates (cm/day) were calculated from a simple linear regression of petiole length against the number of days from the initial measurement using Excel Sp-1. All comparisons between means were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test (MiniTab Version 13) for multiple comparisons. To determine leaf longevity, we selected twenty immature leaves on May 12 and marked them with a garden stake, as before. Leaves were somewhat evenly distributed within a 20- x 20-m2 area. As leaves matured, we classified each leaf as either an aerial or surface form. We then observed each leaf biweekly and recorded the amount of green tissue remaining on it until the leaf was determined to be dead. We defined a leaf as dead when 50% of the lamina was either necrotic or significantly chlorotic. Missing leaf material was included as dead tissue. Twenty additional leaves were marked on both June 8 and July 3, so that we have longevity measures for 59 leaves (one sample was lost) spanning a period of time from May to August. These measures included 28 surface leaves and 31 aerial leaves. Crowding study We examined the influence of vegetational shading (leaf crowding) on the production of aerial leaf forms in summer 2006. In May, we set up six plots in an open area of the pond. Each plot consisted of two concentric circles, one meter and two meters in diameter, that were marked with garden stakes and string. Three of the plots were designated as control plots and were left undisturbed during the course of the experiment. In the other three plots, leaves were selectively removed from the outer ring in an effort to maintain a 50% open water surface throughout the experiment. Laminae were removed by hand at the water’s surface. We were careful not to enter the plots during the pruning process so that we would not cause injury to the rhizomes of the study plants. Twice a week, we recorded the number of surface and aerial leaves appearing within the inner circle for both control and experimental plots. Results Water conditions Temperature, dO2, and dCO2, although highly variable from day to day, consistently oscillated above and below a central mean value during the growing season (Table 1). In contrast, pond depth decreased from approximately 82 cm in May to 14.5 cm in October, whereas ammonia concentration and water pH remained nearly constant. 2008 P.J. Villani and S.A. Etnier 181 Life-history characteristics Across the growing season, immature leaves were produced rapidly at the beginning of the season, but their numbers gradually decreased over the summer (Fig. 2). As leaves matured, they became surface (floating) leaves early in the season, aerial leaves in mid-season, and surface leaves in late season (Fig. 3). Thus, there were two changes in the predominant leaf form during the growing season, from surface to aerial leaves in May–June and from aerial to surface in August–September. Regardless of form, the number of leaves present on a day-to-day basis was fairly stable until late August, when it began to decrease until the end of the growing season (Fig. 2). Immature surface and aerial leaf forms differed in their appearance underwater. When submerged, the laminae of surface leaves were initially tightly curled, but then began to unfold under water until they were Table 1. Pond water quality parameters measured biweekly during the course of the 2006 summer. S.D. = standard deviation, N = sample size. Parameter Seasonal average Range S.D. N Temperature (°C) 22.7 17–28 2.25 28 Depth (cm) 50.1 14.5–82 17.9 73 Dissolved O2 (ppm) 56.9 10–78 23.8 29 Dissolved CO2 (ppm) 62.5 21–94 20.9 29 Ammonia (ppm) 0.23 0.2–0.8 0.02 28 pH 6.55 6.5–7.5 0.20 29 Figure 2. Total numbers of mature (both surface and aerial) and immature water lily leaves produced over the growing season. Six plots (1 m2) were monitored for leaf production two to three times weekly during the 2005 growing season. 182 Northeastern Naturalist Vol. 15, No. 2 completely open as they reached the water’s surface. The laminae of aerial leaves also started tightly curled, but they remained curled as they approached the surface and only opened after they were completely out of the water. Figure 3. Total numbers of aerial and surface leaves produced over the growing season. Six plots (1 m2) were monitored two to three times per week during the 2005 growing season. Figure 4. Growth rates of water lily leaf types over the growing season. Forty-one leaves produced across the growing season were monitored to determine the growth rate of petioles. 2008 P.J. Villani and S.A. Etnier 183 Petiole growth rate was highly variable at a given time during the season; however, the growth rates of all petioles decreased as the season progressed (Fig 4). Since immature leaves mature almost exclusively into surface leaves in the early and late season and into aerial leaves in the mid-season, we compared growth rates of petioles among the different times of the seasons. Early season surface petioles had a mean growth rate of 53 ± 19.5 mm/day (n = 17), while mid-season aerial petioles had a mean growth rate of 37.8 ± 22.5 mm/day (n = 13). Late season surface petioles had a mean growth rate of 21.6 ± 10.5 mm/day (n = 11). Early and late season surface petioles differed significantly in their growth rates (F2, 38 = 9.59, P < 0.005). To determine whether immature petiole form could be used to predict final leaf form, we compared the maximum diameter of immature petioles on the first measurement date for all leaves. The leaf form of a measured immature petiole was determined retrospectively by following an elongating petiole to maturity. Immature aerial leaf forms had a mean petiole diameter that was larger than surface forms (Table 2). Overall, mature surface and aerial leaves were similar in shape. However, the laminae of surface leaves tend to have a smaller surface area compared to aerial leaves (Table 2; F1, 28 = 2.8, P = 0.11). In addition, the mean longevity of surface leaves was significantly less than the mean longevity of aerial leaves (Table 2). Crowding Among the three control plots with no leaf removal, the mean number of aerial leaves over the course of the study increased steadily, to a maximum of 24. In contrast, only a single aerial leaf was observed during the entire study period in the three experimental plots (Fig. 5). Discussion The natural history of aerial and surface leaves varies with respect to a number of different parameters. Aerial and surface leaves appear at different times during the growing season. While their growth rates are similar, their patterns of growth differ, both during maturation and in their final morphology. We suggest that these differences are due to changing functional and Table 2. Comparison of surface and aerial leaf characteristics in the white water lily. Values are means and standard deviations are given in parentheses. An asterisk denotes a significant difference of P < 0.05. Immature Petiole petiole Lamina Leaf form length (cm) diameter (mm) area (cm2) Longevity (days) Surface 66.95 (11.3) 5.8* (1.4) 339.3 (186.6) 34.6* (7.3) N = 15 N = 12 N = 15 N = 28 Aerial 63.13 (10.4) 7.6* (1.1) 436.6 (126.2) 48.0* (11.1) N =15 N = 32 N = 15 N = 31 184 Northeastern Naturalist Vol. 15, No. 2 physiological demands on the leaf. Lily pads maintain high leaf productivity during most of the growing season (Fig. 2, immature leaves), suggesting that they continually develop new organs that are well suited for the current environmental conditions. The heterophyllic nature of the white lily pad may allow it to optimize its photosynthetic opportunities as natural conditions change with the season. Immature surface and aerial leaves differ in form as they grow. Surface leaves open while still underwater, while aerial leaves remain coiled until they extend into the air. We suggest that the coiled aerial leaves more easily penetrate the canopy of surface leaves already present at the water’s surface. The final morphology of surface and aerial leaves also differs. Compared to surface leaves, aerial leaves have a larger petiole diameter (Etnier and Villani 2007) and tend to have a greater lamina surface area (Table 2). Since aerial leaves occur during mid-season, a larger lamina surface area may allow for greater photosynthetic productivity during the long duration and high-intensity light of summer days. The steady decrease in petiole growth rates across the growing season suggests factors other than leaf type may influence growth. For example, pond depth decreased over the course of our study, thus the amount of petiole growth required to bring a lamina to the surface decreased. Once mature, aerial leaves persist for about 13 days longer than surface leaves Figure 5. The influence of leaf removal on aerial-leaf production in water lily. The production of aerial leaf forms were monitored in six plots, three of which did not have leaves removed from them and three of which had leaves removed randomly to maintain 50% exposure of the water surface. 2008 P.J. Villani and S.A. Etnier 185 (Fig. 3), potentially influencing the seasonal patterns observed in our study. We suggest that aerial leaves are more costly to produce because they require more material, so increased longevity may balance the cost of leaf production. During the growing season, there were two major switches in the predominant leaf form. The switch in the developmental pathway leading to surface or aerial leaves must occur early in leaf maturation. Early in development, when petioles are approximately one-third their final length, immature aerial petioles are already larger in diameter than surface petioles (Table 2). The first switch, from surface to aerial, was relatively gradual and occurred early in the season. The second switch, from aerial back to surface, occurred later in the growing season and was much more abrupt, with a rapid decrease in aerial-leaf abundance. The difference in the rate of these two switching events suggests that the plants may be responding to different stimuli. Factors influencing heterophylly A number of different stimuli have been shown to influence heterophylly in other aquatic species, but these factors are unlikely to be responsible for causing it in the white water lily. Marsilea quadrifolia L. (European waterclover) produces aerial leaf forms when submerged shoots grow out of the water into dry air, a desiccation response mediated by abscisic acid (Lin and Yang 1999, Liu 1984). Desiccation is not likely a stimulus in white water lily because the shoot system remains submerged in persistent ponds or lakes, and both leaf forms are always exposed, at least partially, to the atmosphere. In other species, decreasing water depth favors a switch from submerged to aerial leaf forms (Nohara and Kimura 1997, Titus and Sullivan 2001). In our study, water depth decreased over the course of the growing season. The early switch from surface to aerial leaves occurred while water depth was deepest, while the late season switch from aerial to surface leaves occurred when water depth was shallowest. Therefore, water depth is unlikely the stimulus for heterophylly in lily pads. Low dissolved carbon dioxide and oxygen stimulate a switch from submerged to floating leaves in Nuphar variegata Dur. (Titus and Sullivan 2001). Although the shoot in water lily is under water throughout the growing season, the mature leaf forms of water lily are always exposed to the atmosphere. Furthermore, lily pads have a ventilation system which forces air through the leaves down to the rhizome and roots (Dacey 1981); thus, dCO2 and dO2 are probably not limiting factors in this species. Based on the results of the leaf-removal experiment, we hypothesize that changes in the underwater light environment may stimulate the production of aerial leaf forms. The appearance of aerial leaves coincided with the time of maximum surface-leaf production, when surface leaves completely covered the pond surface. The leaf canopy in terrestrial systems has been shown to alter the quality of the irradiance below the canopy and affect plant growth (Leyser and Day 2003, Smith and Whitelam 1997). With 186 Northeastern Naturalist Vol. 15, No. 2 respect to water lily, a canopy of surface leaves at the water’s surface may affect two aquatic light parameters, namely light intensity (fluence rate) and quality, and both have been shown to influence heterophylly in other species (Goliber 1989, Leyser and Day 2003, Lin and Yang 1999, Schmidt and Millington 1968). The results of our leaf-removal study suggest that maintaining a degree of open water surface, thus potentially allowing natural light irradiance to penetrate down to the rhizomes, significantly repressed the appearance of aerial leaf forms (Figs. 1 and 5). This observation also indicates that the site of stimulus perception is likely the shoot and/or developing leaves. The slow gradual stimulation of aerial leaf forms is likely associated with the increasing pond coverage by surface leaves, which may affect the red/far-red ratio of light (Bodkin et al. 1980). This mechanism, which is likely a phytochrome-mediated response, would produce an adaptive leaf form irrespective of what is causing the change in the light quality, either self shading or shading from other species (e.g., other floating aquatic organisms such as algae or aquatic ferns). The above hypothesis addresses the first switch in leaf form from surface to aerial leaves. However, the second abrupt switch, when aerial leaf production reverted back to surface leaf production, suggests that the plants are responding to a different stimulus. Changes in seasonal photoperiods induce changes in leaf form in some aquatic species (Cook 1969, Kane and Albert 1987, Wallenstein and Albert 1962). Therefore, we hypothesize that the second change in leaf form, which occurred during the long days of summer, is a response to changing seasonal photoperiods. Future studies on heterophylly in white water lily should include the direct manipulation of light parameters under controlled conditions and further study of the effect of photoperiods. While our studies suggests that crowding changes the light environment and thus influences aerial leaf production, crowding may also influence other environmental parameters affecting leaf development. To our knowledge, these parameters have not been addressed in white water lily. Heterophylly in the white water lily may be a response to changes in the light environment. We suggest that the switch from surface to aerial leaf forms early in the season allows the plant to maintain a high photosynthetic rate by placing aerial leaves above the existing leaf canopy. Interestingly, this response is not necessarily simple competition for light between different plants, as a given shoot can be shaded by its own leaves, other lily pad leaves, or even other vegetation. Potentially, this heterophyllic response allows a given plant to maximize its photosynthetic capabilities at a given time in the season regardless of the source of shading. As the surface leaves reach senescence, they are replaced by aerial leaves that are fully exposed to the sun. Late in the growing season, the ambient light levels begin to decrease and the shoot switches back to producing surface leaves. One potential benefit of reverting back to surface leaves is to allow the plant to 2008 P.J. 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