Aliso: A Journal of Systematic and Evolutionary Botany Volume 7 | Issue 3 Article 5 1971 Comparative Serology of the Order Nymphaeales. II. Relationships of Nymphaeaceae and Nelumbonaceae Jean-Pierre Simon Rancho Santa Ana Botanic Garden Follow this and additional works at: http://scholarship.claremont.edu/aliso Part of the Botany Commons Recommended Citation Simon, Jean-Pierre (1971) "Comparative Serology of the Order Nymphaeales. II. Relationships of Nymphaeaceae and Nelumbonaceae," Aliso: A Journal of Systematic and Evolutionary Botany: Vol. 7: Iss. 3, Article 5. Available at: http://scholarship.claremont.edu/aliso/vol7/iss3/5 ALISO VoL. 7, No. 3, pp. 325-350 APRIL 22, 1971 COMPARATIVE SEROLOGY OF THE ORDER NYMPHAEALES II. RELATIONSHIPS OF NYMPHAEACEAE AND NELUMBONACEAE. JEAN-PIERRE SIMON Rancho Santa Ana Botanic Garden Claremont, California 91711 INTRODUCTION In a continuing effort to elucidate the serological reiationships of taxa of the aquatic order Nymphaeales, I am reporting here additiona,l data ob­ ta,ined from cross-reactions with antiser-a produced against species of Nu­ phar, Nymphaea, Victoria and Euryale. An earlier study established that Nelumbo wias serologically isolated from the remaining species of the Order ( Simon, 1970). In addition, the data showed that N elumbo had serological affinities with members of the Magnolia.Jes, Ranuncu1ales and Papaverales which were of the same magnitude to those found between N elumbo and the remaining taxa of the Nymphaeales. MATERIALS AND METHODS Extracts of representative species of Nymphaeales, Magnoliales, Ranun­ cu1ales and other orders of Angiosperms, as listed in Tables 2 and 3 of Simon ( 1970), were inves•tigated. Seeds of additional species of these and other orders of Angiosperms have since been available for this study ( Table 3 and text). The prepar-ation of antigen extracts, antisera and the serological techniques were as described by Simon ( 1970) with some modrifioations a,s described below. In order to save reactants, preliminary comparative tests were carried out using the microimmunodiffusion technique ( Crowle, 1961, p. 222). Those extracts giving positive reactions were reinvestigated using the standard ID method described earlier. Three characteristics of antigen extracts of Nymphaeales taxa ( with the exception of Nelumbo) need to be stressed: 1.-The protein content of the extracts was very low. Under the described conditions of extraction and before standardization ( Simon, 1970), the pro­ tein content of delipified seed extracts of the taxa ranged from 0.20-0.35 mg/ml, as compared to 1.6-2.4 mg/ml obtained with Nelumbo under com­ parable extraction techniques. 2.-The antigen extracts were rather unstable when stored at -20 C. A heavy precipitate occurred upon defrosting of the extracts stored for as little [325] 326 ALISO [VoL. 7, No. 3 ~ ~ \fg) ~ ···~ .... ·······.... ~ E ~ ® ... ... ... ........... ®E ........... .... 2 @0 @0 @ @I ·· ·· ···· ··· ·· ·· .. ...... ... ..... u @A ® .... \ __ \, Gr)I y .. .... ........... .. / @u @E -····· ··· ···· 4 @0 3 @I @I ©I ~ ~ \iv ~ ~ ~ 6 ~ 5 ~ APRIL 22, 1971] NYMPHAEALES 327 as two weeks. The addition of sucrose ( Heitefuss et al. , 1959), 10% w Iv, to the dialyzed extracts increased the solubility and stability of the extracts up to a period of storage of two months without affecting ID or IE spectra. Addihional length of storage was still detrimental under these oondirtions. Buffers of higher molarity and/or higher pH did not improve the stabHity of the extracts but affected the sharpness of the precipitin bands in ID tests. Extracts containing 10% sucrose were produced for this study and were used within two weeks following preparation. 3.-Antibodies were not readily elicited with the antigen extracts; while s·trong antisera were produced with Nelumbo extracts after only three series of injections ( oa. 60 mg protein, Simon, 1970), a longer series of injections ranging from 7-10 ( ca. 100-130 mg protein) was needed before a stabiliza­ tion of the number of precipitin bands in ID tests was ob1'ained with antisera produced against Nuphar luteum spp. polysepalum, Nymphaea gigantea, Euryale ferox and Victoria amazonica. Due to the exitensive tests to be per­ formed, pooled antisera from pairs of rabbits were used in this study. RESULTS CROSS REACTIONS BETWEEN NYMPHAEALES TAXA The serological data involving taxa of Nuphar, Nymphaea, Victoria, Euryale and Nelumbo are presented in Tables 1 and 2 and Figs. 1-16. Antisera produced against Nuphar luteum spp. polysepalum reacted simi­ larly to extracts of the three subspecies of that taxon. Four precipitin bands were ·oommon to the subspecies and a filth very faint band was afso observed in some replicates of two of the three taxa ( Table 1, Figs. 1-3). Species of Nymphaea shared two bands in oommon with Nuphar and one or two addi­ tional partial identity bands ( type III of Ouchterlony, 1964) were also seen in the ID plates ( Figs. 1, 2). Less serological affinity was shown by species of Victoria since these two species shared only one identity band with Nuphar. Two partial identity bands, and in some replicates one additional very faint band, were also observed in the ID speotra of Victoria amazonica and V. cruziana. Extracts of Euryale and Nelumbo reacted with anti-Nuphar sera giving only partial identity bands. The ID spectrum of Euryale ferox was characterized by two strong and one faint band while both N elumbo nucifera and N. lutea produced only one such band ( Table 1, Figs. 2, 3). IE spectra confirmed the serological s,imilari,ties of the three subspecies of Nuphar luteum ( Fig. 13). At least 8 arcs ( some of them rather faint) located in the anodal region of the IE spectrum were observed in these reactions. Nymphaea gigantea shared three of these arcs as seen in Fig. 13 c, Fig. 1-6. lmmunocliffusion patterns produced in Ouchterlony plates from reactions be­ tween anti-Nuphar luteum ssp. polysepalum ( NU PO Fig. 1-3) or anti-Nymphaea gigantea ( NY GI, Fig. 4-6) sera ( central wells) and antigen ex;traots of Nymphaeales taxa. Key to symbols: NU MA, Nuphar luteum ssp. macrophyllum; NY TE Nymphaea tetragona; NU VA, Nuphar luteum ssp. variegatum; EU FE, Euryale ferox; VI AM, Victoria amazonica; CI CR, Victoria cruziana; NY AL, Nymphaea alba; NE LU, Nelumbo lutea; NE NU, Nelumbo nucifera; NY CA, Nymphaea capensis. 328 ALISO [VoL. 7, No. 3 (vi'\ ~ -A' \t.!!) 7 ~~ e ® ® ®0 I@\ 9 ®E 10 ~ @E - ®L ~ {NE\ ~ ~ ~ \lV II ---~ \iv 12 APRIL 22, 1971] NYMPHAEALES 329 while the IE spectrum of Nymphaea tetragona showed only one arc posi­ tioned further towards the anode ( Fig. 13 d). The positions of the arcs in both Victoria amazonica and Euryale ferox were rather similar and only one arc was observed in reactions involving Nelumbo lutea antigens (Fig. 13 ). Antisera prepared against Nymphaea gigantea produced three distinct precipitin bands in the ID plates when reacted against N. gigantea antigens. One additional very faint band was obs·erved in some replicates but not in others (Table 1, Figs. 4-6). However, other species of Nymphaea reacted giving two ( N. alba, N. capensis) or only one identity band ( N . tetragona) with th:is antiserum ( Figs. 4, 5). Extmcts of subspecies of Nuphar luteum produced two identity bands and the ID tests indicated that these were the product of the same antigens found in Nymphaea extracts. In the case of Nuphar luteum ssp. variegatum the second band was faint and was not observed in aH the replicates. Additi:iona,l faint partial identity bands were also observed in some cases ( Figs. 4, 5). Both, Victoria amazonica and V. cruziana produced one identity and one partial identity rather faint band in the ID tests with anti-Nymphaea serum. Euryale ferox and both Nelumbo species produced only two and one partial idenbity band respectively ( Figs. 5, 6). IE analyses show serological differences among Nymphaea species as in­ dicated by the number •and variable position of the arcs produced in the IE spectra (Fig. 14). Four sharp and one diffuse arcs wern produced in the test reaction. However only two of the former and a more intense anodal arc were observed in the IE spectrum of Nymphaea alba ( Fig. 14 b). The Immunoelectropherogmm of Nymphaea tetragona showed only a major leading anodal prec:ipitin arc and a more diffuse one at the same position ( Fig. 14 c). The IE spectra of both Nuphar luteum ssp. polysepalum and Victoria amazonica were markedly different from those produced by the Nymphaea taxa and from one another (Fig. 14 d-e) . Finally, both Euryale f erox and N elumbo lutea spectra were charactel'ized by the presence of only one faint arc positioned towards the anode. Antisera produced to Victoria amazonica gave five precipitin bands in the test reaction ( Table 2, Figs. 7-9). Victoria cruziana, however, shared only three such bands with the former species but one additional type III band was also present in the ID plates. Extracts of species of Nuphar, Nymphaea and Euryale shared only one band in common with Victoria species (Figs. 7-9, Table 2). With the exception of Nymphaea alba, one additional faint partial identity band was observed in reactions involving Nuphar and Nymphaea anti:igen extracts and anti-Victoria serum. Two such bands were present in reactions with Eury ale f erox and N elumbo antigen extracts and in the latter mse one of these type III bands was very faint ( Figs. 8, 9). Fig. 7-12. Immunodiffusion patterns produced in Ouchterlony plates from reactions be­ tween anti-Victoria amazonica (VI AM, Fig. 7-9) or anti-Euryale ferox (EU FE, Fig. 10-12) sera ( centra wells) and antigen extracts of Nymphaeales taxa. Symbols as for Fig. 1-6. 330 ALISO [VoL. 7, No. 3 ,»~ ... - ~ ; . ~ ~ ,. ~ -- ~ . --~--;,. - - . - ~~- - -- - -- - . ----- x- ,, ~ ~~· ~- =----- -' . Fig. 13. Immunoelectrophoresis spectra resulting from reactions between anti-Nuphar luteum ssp. polysepalum serum and antigen extracts of Nym­ phaeales: a.