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Evidence for the hybrid origin of Nuphar xrubrodisca (Nymphaeaceae)¹

^a Department of Biology, Southwest Missouri StateUniversity, Springfield, Missouri 65804–0095; ^b Department of Ecology and Evolutionary Biology,University of Connecticut, Storrs, Connecticut 06269–3042;and ^c Department of Plant Biology,University of New Hampshire, Durham, New Hampshire 03824

	ABSTRACT

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Plants intermediate in appearance between Nuphar microphyllaand N. variegata (Nymphaeaceae) have long been assumed to bethe result of hybridization. The evidence for this is based primarilyon field observations of morphology, poor fruit production, closegeographical proximity of presumed parent species, and limited pollensterility data. Fertile populations of the same plants have also beendocumented. We employed multivariate analyses of morphology, pollenfertility studies, and random amplified polymorphic DNA (RAPD) markersto test the hypothesis that Nuphar x rubrodiscarepresents a natural interspecific hybrid between N.microphylla and N. variegata. Examination of 15morphological characters demonstrated the intermediacy of N.x rubrodisca between N. microphylla and N.variegata, and the pollen data revealed a markedly lower meanpollen viability in N. x rubrodisca (23%)compared to the other two species (91 and 86%, respectively). Eight 10-mer primers produced 13 species-specific RAPD markers forN. microphylla and nine for N. variegata, with all 22markers present in N. x rubrodisca. The datafrom RAPDs are concordant with morphology in implicating N.microphylla and N. variegata as parents of N.xrubrodisca.

Key Words: hybridization • morphology • Nuphar • Nymphaeaceae • randomamplified polymorphic DNA (RAPD)

	INTRODUCTION

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Instances of hybridization in aquatic angiosperms remain poorlydocumented, with few studies presenting even basic statistical ormolecular evidence (Les and Philbrick,1993). Hybridization has been investigated in <20%of aquatic angiosperm genera. From this sample, persuasive evidence ofnatural hybridization has been presented for 57% of these genera(Les and Philbrick, 1993). Such studiesare important because documentation of natural hybrids is the first stepto understanding the significance of hybridization in aquaticangiosperms.

Several reports of interspecific hybridization exist forNuphar (Les and Philbrick, 1993). Nuphar species occupy a diversity of freshwater habitatsincluding ponds, lakes, streams, and slow-moving rivers. Nuphar is distributed in temperate regions of North Americafrom Alaska to Newfoundland south to northeastern Mexico and Cuba. Inthe Old World, Nuphar occurs in temperate Eurasia, throughoutEurope south to northern Africa, west to the Kamchatka Peninsula,Russia, and Japan (Beal, 1956).

Nuphar species are taxonomically difficult. Although themost recently published revision of Nuphar combined all NorthAmerican taxa under the single species name N. lutea (L.) Sm.(Beal, 1956), the present taxonomicreevaluation using morphological and molecular data has failed tosupport this concept (Padgett, Les, and Crow,1996). For instance, two North American species with greatlyoverlapping ranges, N. variegata Durand and N.microphylla (Pers.) Fern., are readily distinguishable. Nupharvariegata has more sepals, a greater number of stigmatic rays,larger fruits, longer anthers, and is generally larger overall thanN. microphylla (Fassett,1957; Voss, 1985).

The conspicuous differences of these species led early taxonomists tosuspect the occurrence of hybridization between the two following thediscovery of what they regarded as morphologically intermediatespecimens. Peck (1881) named Nupharadvena (Ait.) Ait. f. var. hybrida Peck from plantsintermediate in morphology between N. variegata and N.microphylla and suggested its possible hybrid origin from thesespecies. Fletcher (1881) similarlyregarded intermediate plants with poorly developed fruits near Ottawa,Canada, as putative hybrids between N. variegata and N.microphylla. Specimens sent to R. Caspary by Fletcher also weredetermined as hybrids between N. variegata and N.microphylla because of their apparent morphological intermediacyand deformed pollen grains (Fletcher,1882, 1883; Macoun, 1883). Other intermediate plantslacking fruit development and with poorly developed pollen were laterobserved growing in the proximity of N. variegata and N.microphylla in the Adirondacks of New York (Morong, 1886).

However, several Vermont populations were known that were similar inappearance to the putative hybrids, but possessed well-developed fruitsand viable pollen. Furthermore, they occurred at a considerabledistance from populations of either presumed parental species (Morong, 1886). Morong(1886) described these fertile plants as a distinct species,N. rubrodisca Morong. Peck(1899) later elevated his N. advena var.hybrida to species status (as Nymphaea hybrida (Peck)Peck ). Others, however, retained the hybrid status (e.g.,Nymphaea x fletcheri Lawson) for sterilespecimens (Lawson, 1888). (Note thatthe genus name Nymphaea L. was applied to Nuphar priorto the conservation of the latter generic name.) Gray (1895) treated all putative hybrids ofNuphar variegata and N. microphylla in North Americaas Nuphar advena var. minus Morong. He regarded thisvariety as a partially to fully fertile "established hybrid"possibly introgressing with the parental species. Miller and Standley (1912) rejected the hybridorigin of Nuphar rubrodisca, a taxon that they believed to be adistinct species with low fertility that was compensated by asexualreproduction. They suggested that more evidence was necessary before ahybrid origin of N. rubrodisca could be adequatelydemonstrated. Contemporary taxonomists continue to differ on whetherthese plants should be recognized as a distinct species (Hellquist and Crow, 1984; Wiersema and Hellquist, 1997) or merely ashybrids with no distinct nomenclatural status (Voss, 1985; Gleason andCronquist, 1991).

The present study was undertaken to reevaluate the taxonomic statusof Nuphar x rubrodisca by testing the hypothesisof its hybrid origin and parentage. This was done by (1) examining thegeographical distribution of the taxon in comparison to those of theputative parents N. variegata and N. microphylla, (2)using uni- and multivariate statistics to quantify and evaluate thesuggested morphological intermediacy of this taxon with respect to itsputative parents, (3) evaluating the fertility of N. xrubrodisca from pollen stainability data, and (4) usingmolecular markers to ascertain whether N. xrubrodisca exhibits additivity of genetic markers that areunique to each of the putative parental species. It was anticipatedthat the compilation of these data should provide suitable informationto determine whether Nuphar x rubrodisca is ahybrid between N. variegata and N. microphylla, or aspecies distinct from both.

	MATERIALS AND METHODS

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Geographical distributions
Geographical distributions of Nuphar variegata, N.microphylla, and N. x rubrodisca were determinedfrom 281 specimens examined from 15 herbaria (BM, DAO, FLAS, IA, MT, NASC, NHA, NCSC, P, TUFT, UC, UNA, US, V, VT). The geographicallocality of each specimen was plotted on North American base maps toobtain estimates of the distribution ranges for each taxon (see Appendix1 for citation of representative specimens).

Morphological analysis
Morphological data were obtained from 216 of the herbarium specimensexamined for geographical distributions (Appendix 1). Five vegetativeand ten reproductive characters were scored for 77 operational taxonomicunits (OTUs) of Nuphar microphylla, 69 OTUs of N.x rubrodisca, and 70 OTUs of N. variegata. Foreach taxon, means and standard deviations were calculated for allvariables using SYSTAT (version 5.0) software (Wilkinson, 1990). Character means werecompared among the three taxa using an analysis of variance (ANOVA) andwere evaluated for significant differences by performing a Tukey HSDpost hoc test. Data were then arranged in a rectangular matrix forinput in principal components analyses (PCA). Unscorable data weretreated as missing. The matrix included OTUs of N.microphylla, N. variegata, and N. xrubrodisca (216 OTUs x 15 characters; 52% missingdata). The PCA was performed using NTSYS-pc (version 1.80) software(Rohlf, 1993). Data were standardizedby dividing the difference of each variable and its mean by the standarddeviation. Product moment correlations were computed among thestandardized variables, the first three principal component axes wereextracted from the correlation matrix, and OTUs were projected upon eachaxis. Results of the PCA were depicted as a scatterplot representingthe superimposition of components I and II. The percentage variationexplained by each eigenvalue and correlations of variables witheigenvectors were tabulated.

Pollen viability analysis
Pollen viability from 30 accessions (ten of each taxon) was estimatedfrom the percentage stainability of 100+ randomly selected grainstaken from herbarium specimens (Appendix 2). Anthers were removed fromherbarium sheets and dissected in aniline blue/lactophenol followingRadford et al. (1974). Means(percentage viability) and standard deviations were calculated as aboveusing SYSTAT. Differences among means were determined by ANOVA andTukey tests as described above.

RAPD analysis
Total genomic DNA was extracted from young, submersed leaf tissuerepresenting three accesssions of Nuphar microphylla, fiveaccessions of N. x rubrodisca, and fiveaccessions of N. variegata (Appendix 3) using a modified CTABprocedure (Doyle and Doyle, 1987). Amplifications were carried out in 25-µL reactions consisting of 10mmol/L Tris-HCL (pH 8.3), 50 mmol/L KCl, 0.005% Tween 20,0.005% NP-40, 2.0 mmol/L MgCl₂, 100 µmol/L each ofdATP, dCTP, dGTP, and dTTP, 15 ng of primer, 1 µL (~20 ng) DNA,and 0.6 units of AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk,Connecticut). Eight random 10-mer oligodeoxynucleotide primers (OPF-1,OPF-2, OPF-3, OPF-4, OPF-5, OPF-6, OPF-8, OPF-10; Operon Technologies,Alameda, California) were used to amplify DNAs (each reaction used asingle primer). A thermocycle profile of 1 min at 94°C, 2 min at36°C, and 2 min at 72°C was carried out for 45 cycles followedby a 7-min final extension cycle at 72°C.

Amplification products were separated electrophoretically on1.5% agarose gels in 0.5x tris-borate-EDTA buffer and werevisualized by staining with ethidium bromide. Band sizes were estimatedusing a standard marker consisting of BstE II-digested LambdaDNA. A preliminary screening was conducted that included severaladditional Nuphar species (N. japonica, N.lutea, N. advena, and N. polysepala) to identifyRAPD markers specific for either N. microphylla or N.variegata. Non specific markers, as well as markers that occurredin all three taxa (N. microphylla, N. variegata,N. x rubrodisca), were excluded from theanalysis. RAPD data were summarized as the number of markers shared byN. x rubrodisca and either N.microphylla or N. variegata. Band reproducibility wasverified by comparing several replicated amplifications for each markerscored.

	RESULTS

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Geographical distribution
The geographical distribution of Nuphar variegata is widerthan that of N. microphylla, but the ranges of both speciesbroadly overlap in northeastern North America. Nuphar xrubrodisca occurs almost entirely in the zone of overlapbetween N. variegata and N. microphylla (Fig. 1).

View larger version (71K): [in this window] [in a new window]   Fig. 1. Distribution of Nuphar microphylla (dashed line), N . x rubrodisca (circles), and N. variegata (solid line).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Principal components plot (first two components) of Nuphar microphylla (open circles), N . x rubrodisca (triangles), and N. variegata (solid circles) based on morphological variables.

View larger version (76K): [in this window] [in a new window]   Fig. 3. Amplified bands of Nuphar DNA with three different 10-mer primers. Lanes C, F, and I are N. variegata (DNA templates from Padgett 491 , 488 , and 491 , respectively). Lanes B, E, and H are N . x rubrodisca (Padgett 481 , 481 , and 479 , respectively). Lanes A, D, and G are N. microphylla (Padgett 397 ). Refer to Appendix 3 for information regarding collection numbers. Bands in lanes A-C were produced from primer OPF-4, lanes D-F from primer OPF-3, and G-I from primer OPF-2. Arrows indicate species-specific bands of either N. variegata or N. microphylla present in N . x rubrodisca .

	DISCUSSION

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The distribution of Nuphar x rubrodiscasatisfies the first criterion of occupying a zone of parental sympatry. Except for three accessions, N. x rubrodiscaoccurs within the region of overlap of the ranges of N.microphylla and N. variegata (Fig. 1). Populations of N.x rubrodisca apparently do not extend beyond thedistributional limits of either putative parent. All three taxa occupysimilar habitats in lakes, ponds, and sluggish watercourses. Nuphar x rubrodisca often occurs with eitherN. microphylla or N. variegata (occasionally both) inthe same body of water. In Lake Champlain, where all three taxa arecommon, N. microphylla tends to colonize deeper waters withN. variegata in shallow shoreline waters; N. xrubrodisca occurs in depths more or less between the twospecies (D. Padgett, Southwest Missouri State University, personalobservation).

Plants of intermediate vegetative morphology have long been cited asevidence of interspecific hybridization between Nupharmicrophylla and N. variegata, although most reports havebeen anecdotal rather than empirically founded. Even though hybridsoften express parental rather than intermediate characters (Rieseberg, 1995), the numerical evaluation ofboth vegetative and floral features indicated a consistent pattern ofmorphological intermediacy for the majority of traits examined. In aselected set of 15 characters, those of N. xrubrodisca were all quantitatively intermediate, being smallerthan those of N. variegata and larger than those of N.microphylla (Table 1). The morphological intermediacy of N. xrubrodisca is compelling given that both putative parentsrepresent discrete size extremes for these features (Fig. 2). The OTUs of N.x rubrodisca clustered between the three-dimensionalcharacter space of the two putative parents, although were somewhatcloser associated to N. microphylla (Fig. 2). Thus, the statisticalanalyses corroborate what various authors have long suggested, i.e.,N. x rubrodisca is intermediate morphologicallyto N. microphylla and N. variegata. Gottlieb (1972) viewed morphologicalintermediacy as a primary criterion of hybridity.

The morphological intermediacy of Nuphar xrubrodisca is also evident in features (qualitative oroverlapping parental traits) that were excluded from the formalnumerical analysis. Nuphar microphylla typically has fivesepals, whereas N. variegata has six. Not surprisingly,N. x rubrodisca has either five or six sepals,depending on the population. Intermediacy can also be observed inseveral qualitative traits. The stigmatic disk of N.microphylla is dark red, in N. variegata it is yellow, andin N. x rubrodisca it is bright red. Themargins of the stigmatic disk differ among the three taxa in a similarfashion—deeply lobed in N. microphylla, essentiallyentire to undulate in N. variegata, and crenate in N.x rubrodisca. Additionally, Britton (1901) reported that the number ofstamen rows differed among these taxa, with five to seven rows in N.variegata, three or four rows in N. microphylla, and aboutfive in N. x rubrodisca.

The fertility of Nuphar x rubrodisca isreduced markedly in comparison to both N. microphylla andN. variegata. The pollen viability of both putative parentalspecies did not differ significantly, and did not fall below 69%(Table 3). Pollenviability of N. x rubrodisca ranged from 13 to50%, but was significantly lower than the mean of either putativeparent (Table 3). Pollenstainability data indicate that N. x rubrodiscasatisfies the criterion of "partial fertility", but alsothat fertility may be retained at fairly high levels in some instances. Variable fertility among populations of N. xrubrodisca has been observed previously (Morong, 1886). Although low pollen fertility isevident in many populations of N. x rubrodisca,little quantitative data on fruit production or seed viability exist. Morong (1886) found only a single fruitwith two or three seeds in a survey of New York populations ofN. x rubrodisca. In Vermont, however, he foundan abundance of fruit, but gave no indication of seed number (Morong, 1886). Four fruits collected recentlyfrom Vermont contained only five, six, eight, and nine seeds each, withnumerous undeveloped ovules (D. Padgett, Southwest Missouri StateUniversity, unpublished data). Compared to the numerous seeds typicallyfound on herbarium specimens of N. microphylla and N.variegata, the level of seed set in N. xrubrodisca appears to be extremely low. Some herbariumspecimens of N. x rubrodisca also contain fruitswith numerous, well-developed seeds, although their viability remains tobe demonstrated.

RAPD data clearly indicated molecular additivity in Nupharx rubrodisca. The putative parental species N.microphylla and N. variegata each possessed several uniqueRAPD markers from a survey of eight primers (Table 4). The surveyed plants ofN. x rubrodisca combined all 22 markers thatdistinguished the putative parental species (Table 4; Fig. 3). Because a wider survey ofother Nuphar species indicated that these genetic markers wereapparently restricted to the two putative parental species, it isdifficult to accept any other explanation for their shared presence inN. x rubrodisca other than as a result ofhybridization. Thus, the RAPD data provide compelling evidence thatN. x rubrodisca indeed represents an interspecifichybrid of N. microphylla and N. variegata.

All four criteria of hybridity that were addressed in this study havebeen demonstrated. Although other criteria remain untested (e.g.,synthetic hybrid production), we believe that the evidence presenteddemonstrates a high degree of confidence for the hybrid origin ofNuphar x rubrodisca. Additional features ofN. x rubrodisca are also consistent with thisinterpretation. Nuphar x rubrodisca isfrequently found in the same body of water as N. microphyllaand/or N. variegata, and these are most likelyinsect-pollinated based on floral studies of other related species(Schneider and Moore, 1977; Ervik, Renner, and Johanson, 1995; Lippok and Renner, 1997). All three specieshave bisexual flowers and are likely to be outcrossing. Like allNuphar species, N. x rubrodisca isstrongly rhizomatous, which would allow for an almost indefiniteperpetuation of sterile hybrid offspring. The chromosome number(2n = 34) of both parental species (and for allNuphar species examined) is identical and constant (Les and Philbrick, 1993). All of these factors canbe viewed as conditions that would not deter hybridization.

Although the present evidence strongly suggests that Nupharx rubrodisca is a hybrid derived from N.microphylla and N. variegata, it is difficult to determinewhether this taxon should be recognized as a discrete hybrid species. There is some evidence that would support the discrete hybrid speciesstatus of N. x rubrodisca. Nuphar xrubrodisca is distinct morphologically from N.microphylla and N. variegata, at least for 13 of thecharacters evaluated statistically (Table 1). Pollen fertility and seedproduction are high in some populations of N. xrubrodisca. The presence of N. xrubrodisca in localities where neither parent occurs indicatesthat some effective dispersal and establishment of new populations ispossible (although extirpation of the parental species cannot be ruledout in such instances). Nuphar x rubrodiscaproliferates vegetatively, and its establishment within aquatic systemsmay be the result of drifting rhizome fragments. Additionally,waterfowl transport of small rhizomes may also take place. It remainsto be demonstrated whether N. x rubrodisca canpropagate sexually through self-fertilization.

We have not observed any evidence to indicate that Nupharx rubrodisca has diverged from either N.microphylla or N. variegata. Morphology and RAPD markersshow intermediacy or additivity rather than any features unique toN. x rubrodisca that might indicate the presenceof a functional isolating barrier between it and the other two species. Instead, the observations presented strongly suggest that hybridsbetween N. microphylla and N. variegata may occurrepeatedly, and that N. x rubrodisca does notappear to represent a stabilized hybrid or a monophyletic assemblagederived from a single ancestral hybridization event. In accordance withthis interpretation, we designate these hybrids nomenclaturally asN. x rubrodisca, a "nothospecies"(Greuter et al., 1994).

Evidence from phylogenetic analyses has been shown to increase thesuspicion and/or detection of the hybrid nature of taxa (Rieseberg and Brunsfeld, 1992; Rieseberg, 1995). As part of a largerphylogenetic study of Nuphar, parsimony analyses ofmorphological and molecular data have indicated a New World/Old Worlddivergence largely congruent with current geographical distributions(Padgett, 1996, 1997). Among the two lineages, N.microphylla is positioned within the Old World lineage and N.variegata within the New World lineage (Figs. 4–6). Analyses of nuclearand chloroplast DNA sequences have offered different alliances forN. x rubrodisca with either lineage. Analysisof nuclear DNA places the N. x rubrodisca in the"New World" clade with N. variegata (Fig. 4), while chloroplast DNApositions this taxon in the "Old World" clade with N.microphylla (Fig. 5). Thephylogenetic position of N. x rubrodisca in acombined analysis is as a sister taxon to the "New World"lineage (Fig. 6). Thediscordance between the independent phylogenies favors the hybrid originof N. x rubrodisca and illustrates the effect ofhybridization on phylogeny. This outcome also emphasizes the need tosurvey both nuclear and organellar genomes in phylogenetic studies(Swofford et al., 1996).

View larger version (23K): [in this window] [in a new window]   Figs. 4–6.  Simplified phylogenetic trees of Nuphar and placement of N . x rubrodisca . 4. Tree derived from nucleotide sequence data of internal transcribed spacer regions (nrDNA). 5. Tree derived from nucleotide sequence data of matK gene (cpDNA). 6. Tree derived from combined nuclear and chloroplast data.

Nuphar x interfluitans Fern. was described byFernald (1942) as a hybrid betweenN. advena and N. sagittifolia Walt. It also displaysmorphological intermediacy, is highly sterile (lacks fruits), andoccurs within the proximity of the putative parents. ExperimentalF₁ hybrids between N. advena and N.sagittifolia yielded only 17.4% fruit set and poor seedlingviability (DePoe and Beal, 1969). Morestudy of the plants from the localities where N. sagittifoliaand N. advena overlap is needed to permit a more accurate interpretation of this putative hybrid. The hybrids described in eachof these instances appear to represent spontaneous F₁ plantssuch as those that we recognize as N. xrubrodisca. All have also been designated nomenclaturally asnothospecies rather than as stabilized hybrid species.

Additional Nuphar hybrids are suspected (e.g., N.variegata x N. polysepala; N. variegatax N. advena), but these have not been studied in anydetail (Wood, 1959; Brayshaw, 1993). Nuphar oguraensisMiki var. saijoensis Shimoda (Shimoda,1991) was initially perceived to be a hybrid between N.japonica and N. oguraensis (M. Shimoda, Towa Kagaku Co.,personal communication) and deserves renewed study. Likewise, theoccurrence of a number of taxonomically "difficult"intermediate plants have suggested the possibility of hybridizationbetween N. oguraensis and both N. japonica and N.subintegerrimum (Casp.) Makino (Y. Kadono, Kobe University,personal communication). A more detailed evaluation of these complexesmay turn up further evidence of hybridization in Nuphar.

Hybridization may occur frequently in Nuphar. Here we haveprovided evidence to support the interpretation of N. xrubrodisca as a hybrid nothospecies that spontaneously resultsfrom the natural crossing of N. microphylla and N.variegata. Most other putative Nuphar hybrids that havebeen studied in any detail display similar characteristics. Ourconclusions fail to corroborate Miller andStandley (1912) who did not accept the hybrid origin ofN. x rubrodisca. Multivariate analyses indicatethat flower size and the number of leaf veins are the most effectivecharacters for separating N. microphylla, N.variegata, and N. x rubrodisca, with fruitsize and leaf sinus length of secondary importance (Table2).

	FOOTNOTES

 
¹ The authors thank B. Hellquist for his valuable field assistance; L. Bogle for advice regarding the pollen analysis; J. Gabel for assistance with the RAPD analysis; N. Miller for providing literature of C. H. Peck; M. Shimoda for information on Japanese Nuphar ; J. Sullivan, L. Bogle, T. Philbrick, and T. Lee for critical reading of an earlier draft of the manuscript. The International Water Lily Society and the New Hampshire Agricultural Experiment Station are gratefully acknowledged for funding portions of this work. This study represents part of a dissertation by the senior author conducted under the direction of G. Crow in partial fulfillment of the doctoral degree at the University of New Hampshire. This paper is Scientific Contribution Number 1975 from the New Hampshire Agricultural Experiment Station.

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