American Journal of Botany 87(6): 898±902. 2000.

BRIEF COMMUNICATION

POLLINATION BY FLIES, BEES, AND OF NUPHAR OZARKANA AND N. ADVENA ()1

BARBARA LIPPOK,2 ANGELA A. GARDINE,3 PAULA S. WILLIAMSON,3 AND S. S. RENNER4,5

2An der Windkunst 2, D-52134 Herzogenrath, Germany; 3Department of Biology, Southwest Texas State University, San Marcos, Texas 78666 USA; and 4Department of Biology, University of Missouri-St. Louis, St. Louis, Missouri 63121 USA and The Missouri Botanical Garden, St. Louis, Missouri 63166 USA

Nuphar comprises 13 species of aquatic perennials distributed in the temperate Northern Hemisphere. The European species N. lutea and N. pumila in Norway, the Netherlands, and Germany are pollinated by bees and ¯ies, including apparent Nuphar specialists. This contrasts with reports of predominant pollination in American N. advena and N. polysepala. We studied pollination in N. ozarkana in Missouri and N. advena in Texas to assess whether (1) there is evidence of pollinator shifts associated with ¯oral-morphological differences between Old World and New World species as hypothesized by Padgett, Les, and Crow (American Journal of Botany 86: 1316±1324. 1999) and (2) whether beetle pollination charac- terizes American species of Nuphar. Ninety-seven and 67% of ¯ower visits in the two species were by sweat bees, especially Lasioglossum (Evylaeus) nelumbonis. Syrphid ¯y species visiting both species were Paragus sp., Chalcosyrphus metallicus, and Toxomerus geminatus. The long-horned Donacia piscatrix was common on leaves and stems of N. ozarkana but rarely visited ¯owers. Fifteen percent of visits to N. advena ¯owers were by D. piscatrix and D. texana. The beetles' role as pollinators was investigated experimentally by placing ¯oating mesh cages that excluded ¯ies and bees over N. advena buds about to open and adding beetles. Beetles visited 40% of the ¯owers in cages, and ¯owers that received visits had 69% seed set, likely due to beetle-mediated geitonogamy of 1st-d ¯owers. Experimentally outcrossed 1st-d ¯owers had 62% seed set, and open-pollinated ¯owers 76%; 2nd-d selfed or outcrossed ¯owers had low seed sets (9 and 12%, respec- tively). Flowers are strongly protogynous and do not self spontaneously. Flowers shielded from pollinators set no seeds. A comparison of pollinator spectra in the two Old World and three New World Nuphar species studied so far suggests that the relative contribution of ¯ies, bees, and beetles to pollen transfer in any one population depends more on these ' relative abundances (and in the case of Donacia, presence) and alternative food sources than on stamen length differences between Old World and New World pond-lilies.

Key words: bee pollination; beetle pollination; ¯y pollination; Nuphar; Nymphaeaceae; pollinator spectra; self-com- patibility.

The Nuphar, spatterdock or yellow pond-lilies, work summarized therein), N. lutea (L.) Sm. from Europe consists of aquatic perennials distributed throughout the and southern Eurasia (Ervik, Renner, and Johanson, temperate Northern Hemisphere. Molecular studies of the 1995; Lippok and Renner, 1997, and earlier work sum- North American and European members of the genus led marized therein), and N. pumila (Timm) DC. from eastern to the recognition of 13 distinct lineages (excluding hy- Scandinavia and northern Eurasia (Lippok and Renner, brids), ®ve in Europe and Asia and eight in North Amer- 1997). A few observations on the northwestern American ica (Padgett, 1997, 1998; Padgett, Les, and Crow, 1999). species N. polysepala Engelmann by V. Grant and B. An earlier assessment (Beal, 1956) that recognized only Meeuse were summarized by Schneider and Moore two species worldwide cannot be upheld (see also Wier- (1977). Below (Results and Discussion) we add new de- sema and Hellquist, 1997). Pollination has been studied tails of Grant's observations on N. polysepala based on in N. advena (Aiton) W. T. Aiton from the southeastern correspondence with him (letters to SSR of 12 and 25 United States (Schneider and Moore, 1977, and earlier August, 1999). These four species appear to have different pollinator 1 Manuscript received 26 October 1999; revision accepted 24 Feb- ruary 2000. spectra, with the European species pollinated by ¯ies and The authors thank S. Hudson for help with plant identi®cation, N. bees, the American species by beetles, with ¯ies and bees Whiteman for information on North American Donacia, and the follow- playing but a secondary role (Schneider and Moore, ing for identifying insects: M. Arduser, St. Louis (bees), S. Lingafelter, 1977; Ervik, Renner, and Johanson, 1995; Lippok and USDA, Beltsville, MD (beetles), and C. Thompson, USDA (¯ies); Renner, 1997). The view that Nuphar as a genus is close- George Yatskievych for help locating study populations in Missouri; T. Arsuf® of Aquarena Center for assisting with logistics at San Marcos; ly adapted for pollination by beetles comes from a study and J. Ott for helping PSW with data analysis. of N. advena in central Texas (Schneider and Moore, 5 Author for correspondence (e-mail: [email protected]). 1977; following then prevailing broad species concepts 898 June 2000] BRIEF COMMUNICATION 899

[Beal, 1956], Schneider and Moore treated N. advena as we wanted to assess whether different ¯oral morpholo- ssp. macrophylla (Small) Beal. of the European species gies of Old and New World species are associated with N. lutea). Schneider and Moore found an average of three different pollinators as suggested by Padgett, Les, and long-horned leaf beetles, Donacia piscatrix Lac., in 32 Crow (1999). These authors have shown that Nuphar N. advena 1st-d (female stage) ¯owers. The beetles car- comprises two major clades. Of the four species whose ried copious amounts of pollen, and Schneider and Moore pollination has been studied, N. advena and N. polysepala judged them more effective pollinators than the sweat belong to the New World clade, and N. pumila and N. bees, honey bees, and ¯ies that also visited the ¯owers lutea to the Old World clade. A ®fth species investigated ``because of [the beetles'] abundance and the length of here, N. ozarkana (G. S. Miller & Standley) Standley, time which they remain in the ¯owers, whereas the bees also represents the New World clade. [Nuphar ozarkana soon departed . . . [ ].'' Whether such an assessment is considered a synonym of N. advena by Wiersema and holds would depend on the plant's mating system, espe- Hellquist (1997), whereas Padgett, Les, and Crow (1999) cially on the long-term effects of sel®ng, but nothing is accord it species status based on relative matK and in- known about genetic neighborhood sizes in Nuphar. ternal transcribed spacer (ITS) sequence divergence Robertson (1889), studying the same species, N. ad- among 13 species and two hybrid lineages of Nuphar.] vena, in Illinois and Florida, found it mainly pollinated All these species have bowl-shaped ¯owers with seem- by sweat bees (Halictus pectoralis) and shore ¯ies (No- ingly easily accessible pollen and nectar rewards. How- tiphila sp.), and he regarded Donacia piscatrix as ``worse ever, New World and Old World ¯owers differ markedly than useless when it comes to pollination.'' in anther lengths and stigmatic disk sizes (Padgett, Les, The fossil record of in North America goes and Crow, 1999). Different-sized reproductive parts or back about 58 million years, with one of the fossils from different amounts of nectar might be differentially attrac- Alberta belonging to subgenus Donacia (Donacia), the tive to ¯ies, bees, or beetles. Given the widely different most derived group (ten Nearctic species and ®ve Old assessments of the role of Donacia beetles as Nuphar World species), which feeds and oviposits exclusively on pollinators (Robertson, 1889; Schneider and Moore, submerged portions of nymphaeaceous leaves, stems, and 1977), we also investigated these beetles' effectiveness peduncles (Hoffman, 1940; Askevold, 1988, 1990, 1991). as pollinators experimentally at the same site as Schnei- Perhaps in¯uenced by the beetles' exclusive dependence der and Moore (1977). on Nymphaeaceae, which in many areas translates as Nu- phar, there has been a tendency to assume reciprocal de- MATERIALS AND METHODS pendence on the part of the ¯owers on the beetles for pollination. Thus, the numerous stamens, ¯at stigmatic Observations on N. ozarkana were made between 4 and 14 July, surfaces, nocturnal ¯ower closure, and intense sweet 1997, in southeastern Missouri, USA at Poplar Bluff (latitude 36Њ43Ј scent of Nuphar ¯ower have been interpreted as ``pri- N, longitude 90Њ23Ј W) along the Beaver Dam Creek, a clear, slow- mary adaptations to assure pollination by beetles,'' and running creek with a maximum depth of 1.30 m. The average water temperature at the study site was 28ЊC. Observations on N. advena were it was suggested that pollination by bees and ¯ies devel- made between 18 and 23 July 1997, near San Marcos, Texas, USA oped later: ``the evolution of other insects (e.g., bees) and (29Њ53Ј N, 97Њ57Ј W) along the Aquarena slough, a streamlet ¯owing other aquatic plants [...]hasbrought about the appearance slowly into Spring Lake of the San Marcos River. The depth of the of new pollinators of Nuphar and the adaptive radiation extremely clear water ranged between 50 and 90 cm, and the average of Donacia'' (Schneider and Moore, 1977). That beetle water temperature during the study period was 25ЊC. Flowers of N. pollination is the ancestral condition in Nuphar, as well ozarkana were observed (by BL) for a total of 22 h over 6 d, usually as being the predominant pollination mode in the genus between 1000 and 1700, and for 2 h during one night. visits to today, became widely accepted in the literature and is a total of 26 ¯owers (1±7/d) were recorded over the entire observation re¯ected in statements such as ``the ¯owers of Nuphar period. Similarly, 26 ¯owers of N. advena were monitored for a total have a close relationship with beetles of the genus Don- of 20 h during 6 d between 1000 and 1900, i.e., from opening to closure, acia, which complete their life cycle in association with and for 2 h during one night. At both sites, stigma and nectary secre- the plant, during which time they facilitate pollination'' tions were tested for glucose with diabetes test paper (Diastix, Bayer, (Schneider and Williamson, 1993). Germany), and intact ¯owers of different ages were tested for terpenoid However, investigations of N. lutea and N. pumila in content indicating ¯oral scent production by immersion in a watery the Netherlands, Norway, and Germany showed that solution of neutral red (Vogel, 1990). these species are pollinated by bees and ¯ies rather than To evaluate the mating system of N. advena we bagged ¯owers just by Donacia or other beetles. The ¯ies include several prior to opening, subjected them to one of the following treatments, and apparent Nuphar specialists, such as the scatophagid Hy- then rebagged them. (1) Flowers were emasculated prior to anther de- dromyza livens and ephydrids of the genera Hydrellia and hiscence to test for the presence of agamospermy. (2) Flower buds were Notiphila (van der Velde, 1986; Ervik, Renner, and Jo- bagged without further manipulation to test for spontaneous sel®ng. (3) First-day ¯owers were emasculated and cross-pollinated with pollen hanson, 1995; Lippok and Renner, 1997). Another spe- from a plant 200 m away. (4) Early 2nd-d ¯owers, still in the receptive cies of Notiphila was found as a N. advena pollinator in stage, were emasculated and cross-pollinated with pollen from a plant Florida (Robertson, 1889). 200 m away. (5) Early 2nd-d ¯owers were self-pollinated with pollen The different role of Donacia beetles in the pollination from their own freshly dehisced anthers. Seed set in naturally pollinated of Nuphar in Europe and (apparently) different parts of ¯owers was quanti®ed for comparison with that in experimental ¯owers. North America led to the present study. By comparing Hand-pollinations were carried out by excising stamens and rubbing beetle, bee, and ¯y abundances and behaviors on two their anthers over the stigmatic disks. Because of the clonal nature of American species with the European ®ndings (Ervik, pond-lilies, widely spaced plants were used to ensure cross-pollination. Renner, and Johanson, 1995; Lippok and Renner, 1997) Bags were removed after anthesis so as not to hinder fruit development. 900 AMERICAN JOURNAL OF BOTANY [Vol. 87

Developing fruits were collected ϳ10 d to 2 wk later and ®xed in 70% min. By contrast, those of N. advena smelled like im- ethanol. Developing seeds and aborted ovules were counted to deter- mature apricots. Fresh petal nectaries of N. ozarkana did mine percentage seed set. not produce visible nectar drops. However, when the Donacia beetles' role as pollinator was tested by placing ¯oating same glucose test strip was touched to all ten nectaries mesh cages over N. advena plants with buds ready to open and adding of a ¯ower it stained weakly. In N. advena, by contrast, D. piscatrix and/or D. texana individuals to each cage; we could not single nectaries produced visible drops that strongly securely distinguish these species in the ®eld. Beetles were inspected stained the glucose test strip. In neither species did the for visible pollen loads, but it is nevertheless possible that they had nectaries stain with neutral red (as also reported by pollen grains from earlier visits to Nuphar ¯owers on them. Because Schneider and Moore, 1977). ¯owers at San Marcos usually had two or three beetles in them, we Flowers of both species opened around 0800, depend- added 2±3 beetles per ¯ower per cage; in total, 30 beetles were added ing on sun intensity. They were functionally female on to ten caged ¯owers. Beetles were placed inside cages, but not directly onto ¯owers, which resulted in some ¯owers being visited by several the ®rst day, because pollen sacs only open on the second beetles, others by none. The cages completely excluded bees and ¯ies. day. First-day ¯owers closed completely between 1800 and 1900, while 2nd- and 3rd-d ¯owers remained par- RESULTS AND DISCUSSION tially open. On the second day, the outer row of stamens bent backwards, presenting pollen, while the mucus on The only species of Nuphar that occurs in Texas is N. the stigmatic rays gradually dried out. During each of the advena (Wiersema and Hellquist, 1997). The entity oc- following three days, one or two rows of anthers matured curring in Missouri is considered a species, N. ozarkana, and presented pollen, while the now completely dry stig- by Padgett, Les, and Crow (1999) based on relative matK ma changed color from dark yellow to brown. Following and ITS sequence divergence among 13 species and two the maturation of the last row of anthers, the petals and hybrid lineages of Nuphar. Based on morphology it was stamens withered, peduncles lost their rigidity, and fruit considered a form of N. advena not worthy of taxonomic ripening proceeded. recognition (Wiersema and Hellquist, 1997). Our mea- Flowers of N. ozarkana were visited by three species surements (below) and ecological observations suggest to of sweat bees, Lasioglossum (Evylaeus) nelumbonis Rob- us that the genetic distinctness of N. ozarkana from N. ertson, Dialictus bruneri (Crawford), and Augochlora advena is paralleled by ¯oral-morphological differences. pura pura (Say), that together made up 97% of all visits. Flowers of N. ozarkana measure ϳ2.5 cm in diameter The syrphid ¯ies Paragus sp., Chalcosyrphus metallicus and are borne on rigid peduncles ϳ2 cm above the water Wiedemann, and Toxomerus geminatus Say together ac- surface. They have six broadly spatulate sepals (N ϭ 9 counted for 3% of the visits. There was a single visit by ¯owers), which are tinged green on the outside and dark a species of Bombus. The bees and syrphids collected or red on the inside, and on average ten petals that are com- ate pollen and occasionally sat on the stigmatic disk to pletely yellow. The ϳ119 stamens and petaloid stamens groom themselves. Pollen-seeking bees predominantly are oblong-linear, 7 mm long, and packed below the stig- visited older, pollen-presenting ¯owers. Donacia pisca- matic disk before anthesis. They open centripetally. As trix Lac. was observed only on 2- or 3-d old ¯owers that is typical of Nymphaeaceae, there is a gradual transition were in late male stage, and of 38 ¯owers checked during from stamens to petals, with petaloid and staminoid in- one night, ®ve contained one beetle each. termediates. The ovary is ovoid and crowned by a cir- Flowers of N. advena at San Marcos received fewer cular ¯attened disk with typically 11 rays of stigmatic insect visits than those of N. ozarkana at Poplar Bluff tissue. There are 25±90 ovules per ovary (N ϭ 8 ovaries), (279 vs. 591 visits to a total of 26 ¯owers studied for and the mature fruits contain on average 41 seeds (N ϭ comparable lengths of time). Sixty-nine percent of all vis- 6 fruits; range 43±72). its were by Lasioglossum (Evylaeus) nelumbonis, the Flowers of N. advena are distinctly larger than those same bee that was the most abundant visitor to Nuphar of N. ozarkana, measuring ϳ3.5 cm in diameter (vs. 2.5 at Poplar Bluff. Syrphids made up 3.9% of all visits and cm in N. ozarkana) and having on average 204 stamens Xylocopa bees 11%. Two species of Donacia were found and petaloid stamens (vs. 119). The sepals are slightly inside the ¯owers, D. piscatrix and D. texana Crotch. obovate, and there are 18 petals (vs. 10 in N. ozarkana; Together they accounted for 15% of all visits to ¯owers N ϭ 5 ¯owers). Red-tinged sepal insides, common in N. during the 20 h of observation. Sometimes, beetles were ozarkana, are not seen in N. advena. The stigmatic disk observed pushing their way through the sepals of N. ad- has 16, rather than 11, rays. On average, there are 327 vena and into a ¯ower before it fully opened, apparently ovules per ovary (vs. 25±90 in N. ozarkana; N ϭ 10 to feed on nectar. Of 22 ¯owers checked during one night ovaries), and the mature fruits contain on average 252 (some being completely closed 1st-d ¯owers, others par- seeds (N ϭ 11 fruits; range 186±353). tially open 2nd- and 3rd-d ¯owers), each contained at First-day (female-stage) ¯owers of both species remain least one beetle, and several contained copulating pairs almost completely closed except for a small triangular (a total of 29 beetles were seen in these ¯owers). At both opening just above the stigmatic disk. The only way for study sites, a few of the outer sepals were visibly gnawed insects to enter these ¯owers and reach the petal nectaries by Donacia. One beetle gnawing on a petal of N. advena is to land on, or crawl over, the stigma, which on the ®rst had pollen attached to its abdomen, and several dissected day is covered with a sticky mucus. The mucus is devoid beetles had abundant N. advena pollen in their guts. of glucose as assayed by diabetes test strips and does not Results of breeding system experiments (Table 1) in- appear to serve as a pollinator reward. Flowers of N. dicate that N. advena is incapable of agamospermy and ozarkana emitted no perceptible odor, even after fresh does not spontaneously self-pollinate. Experimental self- ¯owers had been placed in a clean glass vial for ϳ20 ing of 2nd-d ¯owers yielded 9% seed set; 1st-d ¯owers June 2000] BRIEF COMMUNICATION 901

TABLE 1. Mean seed sets (%) in Nuphar advena in Texas and N. lutea for feeding on plant tissue, including, but not restricted in southwestern Germany after different experimental treatments to to Nymphaeaceae, and for cutting holes into leaf or pe- determine breeding systems. Figures for N. lutea from Lippok and duncle surfaces into which they oviposit, rather than for Renner (1997). feeding on pollen or nectar. However, D. piscatrix and D. Emascu- texana individuals we dissected contained abundant Nu- Treatment Bagged lated N. advena N. lutea phar pollen. We also agree with Schneider and Moore Natural pollination No No 77.5a (11) 64.5a (17) (1977) that beetles appear to be taking up nectar from 1st-day outcrossing Yes Yes 62.3a (10) 60.5a (18) the petal nectaries. In addition to feeding on nectar and 2nd-day outcrossing Yes Yes 12.0b (10) 42.2b (18) pollen, D. piscatrix also mates in Nuphar ¯owers (Marx, 2nd-day sel®ng Yes No 8.6b (16) 50.2b (20) 1957, p.242). Agamospermy Yes Yes 0 (10) 0 (15) Spontaneous sel®ng Yes No 0 (10) 0 (15) Verne Grant (personal communication in Schneider Caged with beetles No No 27.6 (10) n.a. and Moore [1977] and letters to SSR of 12 and 25 Au- Beetle-pollinated1 No No 68.7a (4) n.a. gust, 1999), who studied two populations of N. polyse- 1 Of the ¯owers that opened within the bee-and-¯y-exclusion cages, pala Engelm. on two days in July 1949 near Echo Lake, only four were pollinated by the experimentally introduced Donacia El Dorado County, Sierra Nevada, California, found the beetles; the other ¯owers in the cages set no or few seeds. ¯owers frequented by Donacia beetles, syrphids, and a,b Values with the same letter are not signi®cantly different at the P muscid-like ¯ies; no bee visits were observed. Donacia ϭ 0.05 level (Tukey's test). beetles were abundant on ¯owers in one population, but only one beetle was found in the other population. Bee- tles, syrphids, and muscid-like ¯ies had their venters cov- cannot be autogamously pollinated because of the ¯ow- ered with Nuphar pollen, and since they usually landed ers' strong protogyny. Experimental outcrossing of 2nd- on the stigmas were effective pollinators. d ¯owers resulted in a similarly low seed set (12%), in- The precise role of Donacia as pollinators in nine spe- dicating that stigmas are less receptive on the second day. cies of Nuphar remains to be investigated. The species Experimental cross-pollination of 1st-d ¯owers and nat- occurring in China (N. sinensis Hand.-Mazz.) and Japan ural pollination yielded high seed sets (62 and 78%, re- (N. oguraensis Miki, N. japonica DC., and N. pumila) spectively). Six of ten ¯owers that bloomed inside the have ¯owers very similar to those of N. lutea, and indeed mesh cages with ``beetles-only'' as potential pollinators Donacia gracilipes Jacoby and D. nitidor (Nakane) have set no or few seeds and appeared not to have been visited. been recorded as visitors to the last two species (Kimoto, Four had an average set seed of 69% due to beetle pol- 1981). Of the New World species yet to be studied, N. lination. Since experimental crossing and sel®ng of 2nd- ulvacea (Mill. & Standl.) Standl., N. orbiculata (Small) d ¯owers resulted in much lower seed sets (9 and 12%), Mill. & Standl., N. variegata Durand, and N. sagittifolia the 69% seed set in the cages likely results from beetle- (Walt.) Pursh are similar to N. advena, N. polysepala, and mediated geitonogamous pollination of 1st-d ¯owers N. ozarkana (Padgett, Les, and Crow, 1999). The North (¯owers inside cages belonged to single plants). Taken American N. microphylla (Pers.) Fern. is part of the Old together, these ®gures suggest that natural seed set in N. World clade, and its ¯owers are similar to those of N. advena is mostly due to xenogamy or geitonogamy of pumila. All these species possess petal nectaries and, on 1st-d ¯owers. In European N. lutea and N. pumila, by the second day, abundant pollen, and both rewards are contrast, experimental sel®ng and outcrossing of 2nd-d accessible to ¯ies, bees, and beetles. ¯owers yielded seed sets almost as high as those in 1st- Padgett, Les, and Crow (1999) suggested that the dif- d ¯owers (Lippok and Renner, 1997; Table 1). ferent anther lengths of Old World and New World spe- During the study period, the visitors responsible for cies may in¯uence pollinator effectiveness and selection most pollen transfer between ¯owers in N. ozarkana and (i.e., result in shifts in pollinator spectra). Analyses of N. advena were pollen-collecting sweat bees, especially pollen deposition on pollinators' body surfaces (and sub- Lasioglossum nelumbonis. Schneider and Moore (1977) sequent deposition patterns on stigmas) would be nec- during a 12-mo study of N. advena found halictids (Hal- essary to substantiate a correlation between anther length ictus sp., which may be identical with Lasioglossum nel- and pollinator effectiveness in Nuphar. However, Padgett, umbonis) and honey bees to be the most frequent visitors, Les, and Crow's hypothesis can be assessed preliminarily together with Donacia piscatrix. They observed the bees by comparing pollinator spectra in the two Old World to ``visit more ¯owers in a given amount of time [than and three New World species studied so far (keeping in the Donacia beetles].'' We found Donacia to be infre- mind the unequal and in the case of N. polysepala short quent on N. ozarkana, where they were only seen in 2nd- observation periods). Nuphar lutea and N. pumila (three or 3rd-d ¯owers whose stigmas had dried up and to be populations studied) are mainly visited by ¯ies, with bees abundant on N. advena as earlier observed by Schneider playing a secondary role; N. polysepala (two populations) and Moore (1977). Possibly the larger size of N. advena is visited by ¯ies and Donacia; N. advena (one popula- ¯owers, intense apricot smell, and more copious nectar tion studied in detail) is mainly visited by halictids, ¯ies, (each ¯ower has 18, rather than 10, petals and therefore and Donacia; and N. ozarkana (one population) is mainly nectaries) contribute to that species' higher attractivity for visited by halictids and ¯ies. Flies are thus the only in- beetles. Also, two species of Donacia occur at San Mar- sects visiting all species, while visitation by bees and cos, but only one at Poplar Bluff, and this may explain beetles varies among species and sites (for example, Don- why more beetles were found on San Marcos ¯owers. In acia is absent from N. lutea populations studied in Ger- a monograph of Donacia, Askevold (1990, p.610) reports many, but is an occasional visitor in Norway; Lippok and that D. piscatrix and D. texana have mouthparts adapted Renner, 1997). The relative contribution of ¯ies, bees, 902 AMERICAN JOURNAL OF BOTANY [Vol. 87 and beetles in any one population of Nuphar thus appears North America and Europe. Journal of the Elisha Mitchell Scien- to depend more on these insects' relative abundances ti®c Society 72: 319±346. (and in the case of Donacia, presence) and alternative ERVIK, F., S. RENNER, AND K. A. JOHANSON. 1995. Breeding system and pollination of Nuphar lutea (L.) Smith (Nymphaeaceae) in Norway. food sources than on stamen length differences between Flora 190: 109±113. Old World and New World pond-lilies. HOFFMAN, C. E. 1940. Limnological relationships of some northern An earlier study of N. advena found that bagged ¯ow- Michigan Donaciini (Chrysomelidae: Coleoptera). Transactions of ers produced some seeds (Schneider and Moore, 1977; the American Microscopical Society 59: 259±274. seed set was not quanti®ed), while our bagged ¯owers KIMOTO, S. 1981. New or little known Japanese Donaciinae (Coleop- set no seeds. All species of Nuphar are protogynous, with tera: Chrysomelidae). Bulletin of the Osaka Museum of Natural History 34: 27±46. the only overlap between the female and male stages oc- LIPPOK, B., AND S. S. RENNER. 1997. Pollination of Nuphar (Nym- curring during the morning of the second day when the phaeaceae) in Europe: ¯ies and bees rather than Donacia beetles. stigmatic rays are still receptive (Ervik, Renner, and Jo- Plant Systematics and Evolution 207: 273±283. hanson, 1995; Lippok and Renner, 1997; this report). MARX. E. J. F. 1957. A review of the subgenus Donacia in the Western This would be the time when spontaneous sel®ng might Hemisphere (Colepotera: Donacidae). Bulletin of the American Mu- occur were it not for the position of the stamens below seum of Natural History 112: 191±278. PADGETT, D. J. 1997. Molecular study of yellow pond lilies: the study the stigmatic disk and their extrorse dehiscence, which of Nuphar DNA con®rms, and contradicts relationship theories in together preclude pollen from reaching the stigma unless Nuphar. Water Garden Journal 13: 34±37. via some outside agent. Insect-mediated sel®ng of 2nd-d ÐÐÐ. 1998. Phenetic distinction between the dwarf yellow water- ¯owers is possible because, like other pond-lilies studied lilies: Nuphar microphylla and N. pumila (Nympheaeceae). Cana- (Ervik, Renner, and Johanson, 1995; Lippok and Renner, dian Journal of Botany 76: 1755±1762. 1997; Table 1), N. advena is self-compatible. Whether ÐÐÐ, D. H. LES, AND G. E. CROW. 1999. 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