By: Donald J. Padgett, University of New Hampshire
[Editor’s Note: This article was first published in 1996 in Volume 12-3 of the IWGS Water Garden Journal]
The Study of Nuphar DNA Confirms and Contracts Relationship Theories of Nuphar
The yellow pond lilies (pond lilies, spatterdocks, nuphars) include all the species assigned to the genus Nuphar. These perennial hydrophytes are members of the true waterlily family (Nymphaeaceae) and inhabit freshwater lakes, ponds, and sluggish watercourses. In nature, the yellow pond lilies are distributed in the temperate climate of the Northern Hemisphere. The species range from northeastern Mexico and Cuba to Alaska and Canada in North America, and from northern Europe and throughout Asia to Japan. There are no tropical species of Nuphar.
Admittedly, Nuphar species are not nearly as spectacular as waterlilies. Unlike flowers of true waterlilies (Nymphaea) or lotuses (Nelumbo), that come in an array of colors, Nuphar species produce only yellow flowers, as their common name reveals. Yet, there are species and cultivars with blossoms that are slightly reddened or even orange colored. The flowers are globose, with usually only 5 or 6, rounded, sepals. Also, unlike waterlilies, the petals of Nuphar flowers are seldom noticed, being relatively short and hidden between the larger sepals and numerous stamens. The leaves of yellow pond lilies are primarily floating and thick, ranging from very elongate to more or less heart-shaped. In addition, most species possess a number of thin and almost translucent leaves that remain under water.
Overall, Nuphar plants exhibit an enormous amount of variation in form within any given species. Species of this genus are very similar in appearance, differing primarily in size and number of floral parts and various leaf characteristics. Also, Nuphar species possess the capacity for natural hybridization. This close relatedness among the members, along with their inherent variability, has hindered a clear understanding of evolutionary relationships within this genus. As a consequence, the taxonomic concepts of Nuphar species have been very inconsistent and therefore at times confusing.
Relationship to Waterlilies
The genus Nuphar is considered a member of the waterlily family, the Nymphaeaceae. Its near relative includes Barclaya, Nymphaea, Euryale, Victoria, and Ondinea. It has long been accepted that Nuphar belongs to this family, based primarily on its similarity in form to waterlilies. One taxonomic interpretation, however, once placed Nuphar in its own family (the Nuphacaea), a concept not generally favored. Recent molecular studies have supported the alliance of Nuphar to the waterlily family, and indicate it is most closely related to the genus Barclaya (Les et al 1991). Both of these genera share a relatively remote relationship to Nymphaea, Euryale, and Victoria. The relationship of Nuphar and the Australian genus and the Australian genus Ondinea is unclear.
Classification Among Yellow Waterlilies
The most recent, detailed classification of Nuphar species has remained arguable by many botanists (Beal. 1956). Not conforming to the traditional conviction of numerous Nuphar species, Dr. E. 0. Beal recognized only a single variable species, Nuphar lutea, to occur in North America and Europe. This inclusive species was represented by eight subspecies in North America and two subspecies in Eurasia. Overall, Beal did acknowledge two species. The Far Easter N. japonica from Japan, remained distinct from N. lutea in Seal’s treatment. Prior to Beal’s (1956) study, 17 Nuphar species were proposed for North America alone.
Past systematic studies of Nuphar, like the one of Dr. Beal, have been limited to traditional morphological techniques. Such techniques were used to classify plants solely on the way they physically appear, not necessarily how they may actually be related. Hence, since most Nuphar species are superficially very similar (and appeared to intergrade) to each other, Beal was led to believe that they would be best regarded as one single species with several extreme forms. He designated these forms as subspecies. This enormous variability of form within any (or between) species consequently has rendered Nuphar to be problematic from a taxonomic stand point. Fortunately, there are now many new approaches to analyzing relationships among plant groups where the emphasis is not placed on the physical characteristics of the plants. These techniques have already been very helpful in clarifying certain waterlily relationships. For example, the broad International Waterlily and Water Gardening Society Water Garden Journal P. 13 Vol. 35, No. 1 floating leaves found in both Nelumbo and Victoria by no means imply a close kinship. Instead, this similar leaf form (the so-called “lily pad”) is the result of convergence, where similar features arise in unrelated groups (Les 1993).
Relationships of Yellow Pond Lilies
While systematic classifications of Nuphar species have been relatively numerous, no attempt has been made to explicitly estimate evolutionary positions of the species within this genus. Estimating the evolutionary relationships among Nuphar plants greatly aids in formulating an accurate taxonomic classification (i.e. one that reflects their natural relationships). Thus, the primary objective of my study was to explore the evolutionary relationships among the species of Nuphar from DNA evidence. By comparing them from DNA evidence. By comparing the “nucleotide” sequence of a specific segment of DNA (a gene) between every Nuphar species, a Nuphar genealogy or family tree can be reconstructed. This technique of molecular biology affords an approach in which the reliance on potentially misleading morphological similarities can be circumvented. I chose to examine a gene (known as the ITS region) that occurs in the cell nucleus.
My study of the ITS region of DNA allows us to address several questions regarding the Nuphar relationships. I have used the DNA genealogy to help identify features among Nuphar species that are helpful in indicating their kinship. More specifically, are the elongated leaved plants of N. japonica (of Japan) related to the elongated-leaved plants of N. sagittifolia or N. ulvacea (both of southeastern United States)? Is N. japonica a relative of N. advena (southern U.S.), since both species commonly possess emergent leaves? Are all the so-called “dwarf” species (N. pumila, N. micro, N. pumila, N. microphylla, N. oguraensis) closely related? What species is most closely related to them? The answers to such questions can have practical applications to yellow waterlily growers. Knowing the close relatives of desirable species can indicate what other species will potentially yield successful hybrid offspring if crossed.
Figure l* shows the evolutionary tree that resulted from my molecular analyses. The results of my DNA study provide very strong evidence that there are two major lineages in Nuphar. Interestingly, this relationship is also consistent with morphological evidence, yet it has not been contemplated previously. One lineage contains all the species native to Eurasia, but includes the North American N. microphylla. These species all have five sepals, smooth walled, flagon - shaped fruits with conspicuous necks, and relatively short anthers. The other major lineage consists of all the North American species (except N. microphylla). These taxa all share 6 - 12 sepals, ribbed walled, barrel-shaped fruits lacking necks, and relatively long anthers. The definition of these two groups represents the most important and well supported result of this molecular analysis. These results suggest that features of fruits (overall shape and surface texture), relative anther length, and the number of sepals are good indicators of relationships among Nuphar species.
In the Eurasian lineage, the so-called “dwarf” species (N. pumila, N. microphylla, N. oguraensis, N. pumila, N. microphylla, N. oguraensis) form a natural group, descended from a common ancestor. It is interesting that N. microphylla is restricted to North America, yet bears Old World affinities, surely the result of some western migration at some time in history. The dwarf species appear to be most closely related to N. japonica. Collectively, they all possess lobed, or star-shaped, stigmatic disks. The remote relationship of N. lutea to the other Eurasian species is convincing since this species possesses the unique feature of an entire margined, or round, stigmatic disk. Resolution is much weaker among North American species. Greatest support is found for the basal position of N. polysepala. This is a plausible relationship since N. polysepala is the western representative of the genus In North America and is unique in having 12 sepals. Although N. polysepala overlaps with N. variegata in Alaska, northwestern U.S., and western Canada, this divergent species is relatively isolated and distinct morphologically. The branch maintaining relationships among the remaining North American species is weakly supported and virtually unresolved. The present DNA data are inadequate to elucidate further relationships among these species.
My results clearly indicate that no North American species (except N. microphylla) share any close degree of relationship with Nuphar lutea. Thus, the taxonomic treatment established by Beal (1956), acknowledging but a single species (Nuphar lutea) in Europe and North America is erroneous and it is inaccurate International Waterlily and Water Gardening Society Water Garden Journal P. 14 Vol. 35, No. 1 to apply the name N. lutea to any North American plants. In regards to the questions posed previously our evolutionary tree does provide some answers. The first question was answered above, which stated that fruit shape and sepal number are good indicators of relationships within Nuphar. Although they all share elongated leaves. N. japonica is not closely related to N. sagittifolia nor N. ulvacea, yet the latter two species appear to share a close relationship. Similarly, N. japonica is not related to N. advena, despite the shared characteristic of emergent leaves. As previously assumed, the dwarf species are all very closely related. The closest relative to the dwarfs appears to be N. japonica.
This analysis of DNA represents the first study to propose explicit evolutionary relationships among species of Nuphar. It also constitutes the first taxonomic study of this genus to utilize molecular evidence. While the ITS study remains quite informative, analyses of other genes may offer further information on relationships with Nuphar. I hope to perform similar studies on species of other waterlily genera and continue work on Nuphar utilizing different genes. I have already utilizing different genes. I have already utilized molecular evidence to confirm the hybrid origin and parentage of one North American species.
I wish to thank the International Waterlily Society for funding portions of this study. I am grateful to G. Crow of the University of New Hampshire. D. H. Les of the University of Connecticut, and C. B. Hellquist of North Adams State College for their assistance.
Beal. E. 0. 1956. Taxonomic revision of the genus Nuphar Sm. of North America and Europe. J. Elisha Mitchell Sci. Soc. 72: 317-346.
Les. D. H.. D. K. Garvin and C. F. Wimpee. 1991. Molecular evolutionary history of ancient aquatic angiosperms. Proc. Natl. Acad. Sci. USA 88: 10119-10123.
Les. D. H. 1993. Studies of waterlily relationships: This analysis of DNA represents the first study a search for their roots (or should that be “rhizomes”?) to propose explicit evolutionary relationships among Water Garden Journal 9 (4): 16-22.
**Figure 1 was missing in the original publication, but a likely version of it can be found in this paper:
“Phylogenetic relationships in Nuphar (Nymphaeaceae): Evidence from morphology, chloroplast DNA, and nuclear ribosomal DNA” by Donald J. Padgett, Donald H. Les, and Garrett E. Crow, American Journal of Botany 86(9): 1316 - 1324. 1999.