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3Department of Biology, Southwest Missouri State University, Springfield, Missouri 65804-0095; 4Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, Connecticut 06269-3043; and 5Department of Plant Biology, University of New Hampshire, Durham, New Hampshire 03824
Received for publication November 20, 1998. Accepted for publication February 22, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ITS • matK • morphology • Nuphar • Nymphaeaceae • phylogeny • waterlilies
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Les, Garvin, and Wimpee, 1991;
Moseley, Schneider, and Williamson, 1993;
Les, Schneider, and Padgett, 1997
), supporting a long-established taxonomic tradition (Salisbury, 1806; Caspary, 1891;
Cronquist, 1981
). Recent phylogenetic estimates place Nuphar as most basal in the family and indicate the south Asian genus Barclaya to be its closest living relative (Les et al., in press)
. Interestingly, Nuphar is the only genus in the family lacking tropical species.
Species of Nuphar share several characters that are unique in the Nymphaeaceae and provide evidence for the monophyly of the genus, including a superior gynoecium, abaxial petal nectaries, echinate and anasulcate pollen, emergent fruit maturation, and presence of only a single (outer) satellite vascular bundle in peduncles (Les et al., in press)
. Because of this combination of characters, Kerner (1891)
, Nakai (1943)
, and Takhtajan (1997)
proposed that Nuphar should be recognized as a separate monogeneric family, but this opinion has been largely ignored.
At the intrageneric level, Nuphar is among the most taxonomically troublesome genera in the Nymphaeaceae. Existing treatments (e.g., Morong, 1886;
Harz, 1893;
Schuster, 1907
; Miller and Standley, 1912
; Heslop-Harrison, 1955;
Beal, 1956
) focused mostly on a regional scale and are generally discordant. The most unparalleled classification of the genus was proposed by Beal (1956)
. The great morphological variability, evidence of hybridization, and uniform chromosome number among then-recognized species led Beal (1956)
to treat these 22–25 taxa as one species with nine subspecies. Thus, all Nuphar occurring in both North America and Europe were classified as N. lutea (L.) Sm. In addition to his polymorphic concept of N. lutea, Beal (1955)
recognized a second species, N. japonica DC., from Japan. Although Beal's (1956)
revision met with much dissent (e.g., Sculthorpe, 1967
; Hultén, 1971
; Voss, 1985
; Crow and Hellquist, in press), it remained popular in North America and was adopted in several regional floras and manuals (e.g., Calder and Taylor, 1968
; Correll and Correll, 1972
, Godfrey and Wooten, 1981
; Rhoads and Klein, 1993
). Despite the wealth of systematic attention the genus has received, there have been no specific hypotheses of phylogenetic relationships proposed for Nuphar. Consequently, precise evolutionary relationships within the genus remain enigmatic.
The genus Nuphar is delimited here to comprise 15 taxa recognized tentatively at the rank of species: N. advena (Ait.) Ait. f., N. x intermedia Ledeb., N. japonica DC., N. lutea (L.) Sm., N. microphylla (Pers.) Fern., N. oguraensis Miki, N. orbiculata (Small) Mill. & Standl., N. ozarkana (Mill. & Standl.) Standl., N. polysepala Engelm., N. pumila (Timm) DC., N. x rubrodisca Morong, N. sagittifolia (Walt.) Pursh, N. sinensis Hand.-Mazz., N. ulvacea (Mill. & Standl.) Standl., and N. variegata Durand. These 15 taxa, adopted from generally accepted taxonomies (e.g., Heslop-Harrison, 1955;
Wiersema and Hellquist, 1997
), reflect the complete morphological and geographical variation found within the genus (see Padgett, 1997
). The objectives of this study were to present a comprehensive phylogenetic reconstruction of Nuphar based on three independent data sets (morphology, chloroplast DNA, and nuclear DNA) and to compare and evaluate the resulting hypotheses of phylogenetic relationships.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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SEPAL COLOR (2). In general, the abaxial sepal surface in Nuphar is yellow. The base of the adaxial sepal surface is green in most species, usually progressing to yellow towards the apex. In N. ozarkana and N. variegata the adaxial coloration is red to dark purple.
ANTHER/FILAMENT LENGTH RATIO (3). There are two distinct stamen types found in Nuphar: anthers equaling or longer than the filaments (most North American taxa) and anthers shorter than the filaments (all Eurasian taxa).
ANTHER COLOR (4). The color of the pollen sacs and surrounding connective tissue varies from yellow (most species) to purple (as in N. polysepala).
STIGMATIC DISK MARGIN (5). Stigmatic disks of most Nuphar species have essentially an entire margin. In contrast, several species have a distinctly lobed disk margin.
STIGMATIC DISK COLOR (6). Immature pistils among most Nuphar species have yellow stigmatic disks. When mature, these disks turn green, but remain somewhat yellowish. Dark carmine-colored disks are found among flowers and fruits of N. microphylla.
STIGMATIC DISK SIZE (7). Statistical analysis of Nuphar taxa (Padgett, 1997
) indicates two size classes of stigmatic disk diameter ratios, relative to the diameter of the mature ovary (disk/fruit diameter). These ratio classes are represented by two discrete character states: broad (>0.45) and narrow (<0.45).
FRUIT SHAPE (8). Mature Nuphar are either urceolate or ovoid. Urceolate fruits are usually smaller in size, with a well-defined ovary ("flaggon-shaped" in the sense of Heslop-Harrison [1955]
). Ovoid fruits are larger, and more columnar in appearance.
CONSTRICTION BELOW STIGMATIC DISK (9). Beal (1956)
recognized Eurasian Nuphar lutea taxa as characterized by a strongly constricted style. Morphometric analyses disclose two discrete size classes of the constriction width (Padgett, 1997
) corresponding to either a narrow (2–6 mm) or broad constriction (9–22 mm).
CONSTRICTION/FRUIT DIAMETER RATIO (10). Analysis of constriction widths relative to overall fruit diameters clearly indicates two ratio classes, here treated as discrete character states: constrictions less than one-quarter of the ovary width (<0.25) and constrictions nearly half as wide or greater (>0.40) than the ovary width (Padgett, 1997
). When the constriction below the disk is narrow, the constricted region is usually elongated. This is conspicuous in early fruit maturation with the stigmatic disk raised above the sepals.
FRUIT SURFACE (11). The surface texture of mature ovary walls varies from smooth (as in Nuphar lutea) to vertically ribbed with distinct grooves (e.g., N. variegata). Occasional furrowing on fruits of smooth ovary taxa is subtle and restricted to just below the stigmatic disk.
LEAF HABIT (12). Although all Nuphar species possess submersed basal leaves, exposed leaves are either floating or emergent. Floating lamina occur most commonly in the genus.
LEAF BLADE LENGTH (13). Blade length varies within species and populations, yet several species maintain small-sized leaves when compared to other taxa. The small-leaved taxa are commonly referred to as dwarf Nuphar. Character states (<12 cm or >15 cm) reflect a conspicuous morphological gap distinguishing these small-leaved taxa from larger leaved species (Padgett, 1997
).
LEAF LENGTH/WIDTH RATIO (14). Blade shape was emphasized by Miller and Standley (1912)
and Beal (1956)
to distinguish taxa in Nuphar. Exposed blades can be ovate (1:1.5), orbicular (1:1), or narrowly lanceolate (1:>4).
BLADE SINUS/LENGTH RATIO (15). Basal sinus length relative to total blade length has been used as a reliable diagnostic character in taxonomic keys and descriptions of Nuphar (Beal, 1956
; Wiersema and Hellquist, 1997
). Most species have a basal sinus length that is about one-third of the blade length. Taxa with elongated leaves possess shorter basal lobes and therefore much shallower sinuses. The diminutive taxa exhibit deeper sinuses in comparison to blade lengths.
PETIOLE SHAPE (16). Despite some minor local variation, petiole shape remains constant within Nuphar taxa. Observed cross-sectional shapes of petioles include terete, dorsally flattened ("winged"), trigonous, and dorsally flattened but unwinged variations.
PETIOLE ANATOMY (17). Internal petiole anatomy (lacunar size and arrangement) has been used to distinguish genera (Goleniewska-Furmanowa, 1970
; Chen and Zhang, 1992
) and/or species groups (Conard, 1905
) in the Nymphaeaceae sensu lato. In Nuphar, petiole lacunae are small and arranged in a reticulate fashion, similar to that found in Barclaya. Most taxa possess similarly sized and randomly arranged lacunae. Petioles of N. oguraensis possess a larger, centralized lacuna among the smaller lacunae.
Molecular data sets
Total genomic DNA was isolated from silica gel-dried leaf tissue of 13 field-collected Nuphar taxa using a modified CTAB procedure (Doyle and Doyle, 1987
). No material was available of either N. x intermedia or N. sinensis. Voucher specimens have been deposited at NHA (see Table 4).
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2.5 kb total) were amplified from total genomic DNA using the polymerase chain reaction (PCR) and thermostable DNA polymerase. Primers used for amplification were the trnK-3914F and trnK-2R primers of Johnson and Soltis (1994)
. The double-stranded amplification products were purified by gel isolation in low melting point agarose followed by a secondary GeneClean II purification (Bio101, La Jolla, California). Direct dideoxy sequencing of purified DNAs was performed using Sequenase version 2.0 (United States Biochemical, Cleveland, Ohio) and eight sequencing primers including trnK-3914F, trnK-2R, and matK-1470R of Johnson and Soltis (1994)
and five newly designed primers (Table 3). The matK gene and flanking intron sequences were obtained from 13 Nuphar taxa and from Barclaya longifolia (Table 4).
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. In some taxa, when double-stranded products were difficult to sequence, single-stranded amplifications were performed. The double-stranded amplification products were purified by gel isolation in low melting point agarose followed by a secondary GeneClean II purification. Single-stranded DNAs were purified via centrifugal column dialysis (Baldwin, 1992
). Direct dideoxy sequencing of purified DNAs was performed as in the matK study with four sequencing primers (ITS-2, ITS-3, ITS-4, and ITS-5 according to Baldwin [1992
]). Sequences of the ITS region were obtained from the same 13 Nuphar accessions as in the matK study (Table 4).
Phylogenetic analysis
The boundaries of both the matK gene and the ITS region were determined by comparison to published sequences (Sugita, Shinozaki, and Sugiura, 1985
; Yokota et al., 1989
; Baldwin, 1992
). The phylogenetic significance of the morphology and sequence data was assessed by maximum parsimony methods employing the computer program PAUP, v. 3.1.1 (Swofford, 1993
). All characters were unweighted and unordered. Most-parsimonious trees were found using heuristic searches, with TBR (tree bisection-reconnection) branch swapping, MULPARS, and steepest descent. In the DNA data sets, indels were treated as an alternative character state. Strict consensus trees were constructed from all most-parsimonious trees. Bootstrap analyses (1000 replicates) were conducted to examine the relative level of support for individual clades on the cladograms of each search (Felsenstein, 1985
). Decay indices were used as another measure of the robustness of individual branches (Donoghue et al., 1992
).
Four parsimony analyses were performed: an analysis of morphology, an analysis of matK sequences, an analysis of ITS sequences, and a combined analysis of morphology, matK, and ITS data. The matK and morphology analyses used the closely related Barclaya longifolia and B. rotundifolia, respectively, as outgroups. Initially, representatives of Cabombaceae (Brasenia and Cabomba) and Nymphaeaceae (Nymphaea and Barclaya) were selected as outgroups for the ITS analysis. Partial ITS sequences were obtained for the first three of these genera (available upon request) but could not be readily aligned with any Nuphar ITS sequence. Despite repeated efforts, ITS sequences of Barclaya were not attainable. Thus, the ITS search utilized mid-point rooting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cladistic analysis of the morphological data produced 190 equally most parsimonious trees (Fig. 1), with a length of 32 and CI (consistency index) of 0.84 [CI excluding uninformative characters = 0.82, RI (retention index) = 0.91]. The large number of trees differed mostly within the large clade of Nuphar advena, N. ozarkana, N. variegata, N. ulvacea, N. orbiculata, N. sagittifolia, and N. polysepala. Also, N. microphylla was often aligned as a sister taxon to the remaining dwarf species (N. pumila, N. sinensis, and N. oguraensis).
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nrDNA phylogeny
Among most Nuphar species, 85% (233–235 bp) of the total ITS 1 was determined. Complete ITS 2 sequences of Nuphar measured 242–250 bp in length. Several indels ranging in size from 1 to 5 bp were detected. Partial sequences of 5.8s (61 nucleotides mainly at the 3' end) were obtained for most species and incorporated into the data set. A total of only 252 bp of sequence, mostly of ITS 2, were obtained for N. oguraensis. In N. sagittifolia, 86 (30%) bp were not sequenced from the 3' end of ITS 1. The total number of variable sites between all species was 37, with 16 (43%) of these in ITS 1, 20 (54%) in ITS 2, and 1 (3%) in 5.8s. Potentially phylogenetically informative sites numbered 9 (41%) in ITS 1, 12 (54%) in ITS 2, and 1 (5%) in 5.8s. Mean pairwise distances (as calculated by PAUP) of the ITS sequences between taxa varied from 0.0 to 5.5% within Nuphar (Table 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Although phylogenetic analyses of each data set revealed some well-supported groups of species, relationships among taxa within the New World clade remain poorly resolved. The lack of synapomorphic characters in the morphology data set resulted in a complete polytomy of this group. The matK phylogeny only weakly supported relationships among the largely southern U.S. taxa (Nuphar ozarkana, N. advena, N. orbiculata, and N. ulvacea). Within the New World clade, the matK-based topology did indicate a strong relationship between N. variegata and N. sagittifolia, but failed to elucidate the position of N. polysepala (Fig. 3). The ITS phylogeny placed the northwestern North American N. polysepala at the base of the New World clade, but provided little additional information within this lineage (Fig. 4). Overall, the data sets indicate that the New World taxa are extremely closely related, but distinct from Old World lineage representatives.
Relationships are better resolved within the Old World clade, at least with morphology and ITS data (Figs. 2, 4). From these data sets, the widespread Eurasian Nuphar lutea is resolved at the base of the lineage, followed by the Japanese endemic N. japonica. A monophyletic dwarf Nuphar lineage (N. microphylla, N. pumila, N. oguraensis, and N. sinensis) is revealed by both morphology and ITS data. The matK data offer little phylogenetic information within the Old World clade, except for an indication that N. japonica and N. oguraensis are sister taxa.
Discordance among phylogenies
Discordance between nuclear- and chloroplast-based phylogenies has been detected within several plant groups (e.g., Soltis and Kuzoff, 1995;
Soltis, Johnson, and Looney, 1996
; Kellogg, Appels, and Mason-Gamer, 1996
). Explanations for the cause of incongruence between phylogenetic hypotheses based on cpDNA and other data sets often implicate hybridization and introgression, particularly at lower taxonomic levels in groups noted for interfertility (Doyle, 1992
; Rieseberg and Brunsfield, 1992
; Soltis and Kuzoff, 1995)
. Chloroplast capture via hybridization provides a species with a foreign chloroplast genome, thus profoundly affecting cpDNA-based phylogenetic reconstructions.
Discordance between the independent Nuphar phylogenies is mainly a result of hybridization. The Nymphaeaceae are noted for hybridization (Les and Philbrick, 1993)
and hybridization has indeed been documented in Nuphar (Heslop-Harrison, 1953;
Padgett, Les, and Crow, 1998
). A major difference between the ITS and matK topologies involves the placement of N. x rubrodisca. In the tree based on ITS data, N. x rubrodisca is within the New World clade (Fig. 4), whereas matK data place N. x rubrodisca within the Old World clade (Fig. 3). Nuphar x rubrodisca is a known hybrid taxon between the New World N. variegata and the Old World allied N. microphylla (Padgett et al., 1998
). Identical cpDNA sequences between N. microphylla and N. x rubrodisca indicate chloroplast inheritance from the former. At least from the plants sampled, ITS data identify N. variegata as the pollen donor of the cross (neither N. orbiculata nor N. sagittifolia are potential parents; see Padgett, Les, and Crow, 1998
).
An unexpected result in the matK phylogeny is the close relationship indicated between Nuphar variegata and N. sagittifolia. The alliance of these two taxa is evidenced by three substitutions, which represents the highest level of shared matK sequence variation between any two taxa in the data set. The identical matK sequences of these two taxa is perplexing, since their ranges are widely allopatric. Nuphar variegata is a boreal species in North America occurring mainly north of the glacial boundary, while N. sagittifolia is a coastal plain species limited to Virginia, North Carolina, and South Carolina. Morphologically, the species differ markedly, most obviously by their leaf morphology. To rule out any possibility that DNA contamination might account for this unusual result, several extractions were repeated from different accessions and re-analyzed, always with the same result. Morphology-based or ITS phylogenies fail to elucidate the positions of these two taxa. The cpDNA data suggest that these taxa may have historically occurred in closer proximity where, perhaps, populations of N. sagittifolia had captured the chloroplast genome of N. variegata (or ancestor) following an ancient hybridization event. Nuphar variegata is known to hybridize.
Another unexpected result of the matK phylogeny was the resolution of a clade containing Nuphar japonica and N. oguraensis. This clade is not revealed in the morphological phylogeny. Although both taxa are included in the Old World lineage, N. oguraensis comprises part of the well-supported clade of dwarf species in the morphology cladogram. When the partial ITS sequence of N. oguraensis is included in the analysis, this taxon also is positioned in the dwarf clade. Hybridization as the cause of this discordance between phylogenies is highly tenable. The two species are endemic to Japan and overlap in distribution. Also, interspecific hybridization has been well documented between N. pumila (a dwarf species) and N. lutea, and between the dwarf N. microphylla and N. variegata (Padgett, Les, and Crow, 1998
). Interspecific hybridization involving N. japonica and N. oguraensis has been suggested previously (M. Shimoda, Towa Kagaku Co., personal communication).
Combined phylogeny
The phylogeny based on the combined data set adds internal support for certain lineages and clarifies one of the unexpected matK relationships. Both the strict consensus tree and the majority-rule consensus tree reconcile the phylogenetic position of N. x rubrodisca. In both trees this hybrid taxon is at the base of the remaining New World members. The combined phylogeny likewise places N. oguraensis back in the monophyletic clade of dwarf species with N. microphylla and N. pumila. The association of N. variegata and N. sagittifolia remains highly supported in the combined phylogeny.
Few hypotheses concerning evolutionary relationships within Nuphar have been advanced for comparison. The reconstructed relationships within Nuphar presented here fail to corroborate Beal's (1955, 1956)
hypothesis of a wide-ranging, polymorphic species (as N. lutea) in the New and Old Worlds, that is distinct from N. japonica. All evidence indicates that Beal's N. lutea is not monophyletic, seriously calling into question a taxonomic scheme that has found wide acceptance for decades. Hultén (1971)
remarked on a close relationship of the Eurasian N. lutea sensu stricto with N. polysepala and N. variegata of North America and considered them to form a species complex. A close relationship between these taxa is not supported by either morphological or molecular data, although a relationship between the latter two taxa is evident. The low divergence between Nuphar taxa overall, however, is in agreement with Beal's general concept of closely related lineages within the genus.
The resulting phylogenies estimated herein have an intriguing phytogeographical implication. The analyses of all data place Nuphar microphylla, a boreal North American species, in the same clade as all the Eurasian species. This dwarf species may have migrated from Eurasia via a land bridge following the divergence of the two larger lineages. Without further information, the geographical origin (western Europe or eastern Asia) of the ancestor of this taxon is speculative. The low molecular divergence of N. microphylla from other dwarf taxa supports a more recent dispersal. Given the relatively large size of Nuphar rhizomes and intolerance of seeds to drying or digestion, long-distance dispersal does not seem plausible.
The phylogeny of Nuphar offers a baseline framework to study the evolution of morphological characters. Several floral and fruit features can be evaluated between the two major clades that support two largely geographic groups of species. Species of the Old World group share fruits with elongated necks ("styles" of some authors) and narrow stigmatic disks. Except for N. lutea, the margins of the stigmatic disks of these species are incised to the extent of being lobed. Furthermore, Old World taxa have five sepals per flower and short anthers supported by relatively long filaments (Padgett, 1997
).
In contrast, species of the New World group lack the neck in their fruits and have much broader stigmatic disks that are essentially entire-margined. Flowers of the New World taxa are showier, with more sepals than Old World species (up to 14 in Nuphar polysepala). The anthers in this group are more elongate than those in the Old World group, which possess shorter filament lengths.
The evolution of these reproductive features may be related to certain pollination mechanisms and/or pollinators. Floral biology studies of representative taxa in both clades indicate that all taxa are self-compatible and protogynous, with floral odor and color the primary means of attracting pollinators (Schneider and Moore, 1977
; Ervik, Renner, and Johanson, 1995
). Pollination studies of the Old World Nuphar lutea and N. pumila indicate these species are visited by an array of flies, bees, and beetles but are mainly pollinated by flies, and not by beetles (Van der velde et al., 1978; Ervik, Renner, and Johanson, 1995
; Lippok and Renner, 1997
). Studies of the New World N. advena and N. polysepala reveal pollination predominantly by beetles, but visitation by bees and flies (Schneider and Moore, 1977
, and references therein). Schneider and Moore (1977) asserted that the overall floral structure of Nuphar advena (e.g., broad, flat stigmatic disks, and numerous stamens) assures beetle pollination. It remains to be seen whether the markedly different anther lengths between taxa of the Old and New World groups may influence pollination or pollinator effectiveness or selection.
The lack of phylogenetic information within Nuphar precluded Lippok and Renner (1997)
from hypothesizing ancestral floral features in the genus. Yet, based on floral features, they suggested that the ancestral floral morphology probably favored fly and bee pollination (Lippok and Renner, 1997
).
In conclusion, our study provides insight into the evolution of two major lineages within Nuphar. As illustrated above, the relationships depicted from both morphological and molecular data are in conflict with more recent taxonomic evaluations of the genus. These results provide support for a new classification of Nuphar to accommodate these two phylogenetically distant lineages.
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FOOTNOTES |
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2
Author for correspondence, current address: Department of Biological Sciences, Bridgewater State College, Bridgewater, Massachusetts 02325 (phone, 508 697 1358; Fax, 508 697 1785; e-mail, dpadgett{at}bridgew.edu
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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———, ———, ———, P. S. Soltis, D. E. Soltis, and M. Zanis. In press. Phylogeny, classification and floral evolution of water lilies (Nymphaeaceae; Nymphaeales): a synthesis of non-molecular, rbcL, matK, and 18S rDNA data. Systematic Botany 24: 28–46.
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