Molecular Analysis: A New Look at Umbrella Richard B. Figlar

Taxonomists have long been frustrated in their attempts to decipher the complex evolu- tionary relationships within the genus . Recent molecular research has shed new light on the problem and helped to clarify the long- standing confusion.

The magnolias of section Rytido- spermum-one of sixteen categories that subdivide the 128 of the genus Magnolia-have always been an intensely interesting group, not only for their large and enor- mous whorled , but because several species occur in both eastern Asia and eastern North America. Within the genus, this intercontinen- tal distribution is shared only with section Tuhpastrum, but in that case the two species involved, our native cucumber , Magnolia acumi- nata, and M. liliiflora, the famous Mulan magnolia from China, share few characteristics beyond the same number of chromosomes and the These three closely related magnolras share large, whorled leaves, a to two m and white presence of reduced outer . rangmg from foot feet length, large, flowers with diameters m the six to twelve mches. The The Rytidospermum section, range of flowers, which open after the leaves have developed, are strongly scented, to most according taxonomists, Magnolia tnpetala, above, unpleasantly so. A natme of the consists of six species: MagnoliaAllegheny region of the eastern Umted States, it seldom exceeds tripetala, the umbrella magnolia; M. ;forty feet and is uncommon both m the mld and m cultivation. fraseri, the mountain magnolia; M. At top mght Is the Japanese Magnolia obovata. It grows to macrophylla, the big- magnolia; eighty feet m its native damp, mch, highland forests, and is one of M. obovata (M. hypoleuca), M. the hardiest Asian magnohas (zones 6 to 9). Its shghtly less hardy Chmese M. officinalis var. at bottom also and M. rostrata-the first sister, biloba, right, officinalis, ’ at to ‘grows altitudes from 2,000 5,500 feet, and achieves heights three native to southeastern Umtedup to seventy feet. Its bark is so highly valued as medicine that States into Mexico and the latter the. tree has been nearly extmpated m its native provmces of Hubei three native to eastern Asia, from theand Sichuan 23 24

represent true whorls, the leaves of Rytidospermum magnolias are arranged in false whorls; that is, the individual leaves actually emerge in alternate fashion but with very little stem growth (internodes) between successive leaves. The pattern of many leaves emerg- ing almost simultaneously is called flushmg. Apparently, Rytidosper- mum magnolias adapted this flush- type leaf-emergence pattern in order to compete effectively in the gaps of forest understory during early spring. By producing more leaves more or less simultaneously, such are better able to compete with other species for scarce sunlight. And since little stem growth is produced, the process itself is very energy efficient. Later in the spring, the growth reverts to the more typical pattern, where leaves are produced one at a time along longer stem shoots, as in other magnolias. Flush-type leaf- emergence patterns are common in many other species of the understory; for instance, some of the azaleas, although because of their much smaller leaves, the umbrella effect is less noticeable than in the Rytidosper- mum magnolias.2 Clearly, among the magnolias this Like other members the three within of subgenus Magnolia (one of trait is and for that reason the the M. tend to be red and unique, genus), frmts of tripetala bmght taxonomists have showy They persist for several weeks in late summer. suggested that, despite their intercontinental distri- Kurile Islands and Japan westward to southwest bution, they all form a natural group and should China. Among the various morphological char- be very closely related. This provokes several acteristics shared by members of this group, the questions. Did today’s species evolve from a most distinctive are the enormous whorls of common ancestor? If so, how and when did its deciduous leaves, which are crowded m parasol descendants cross the Pacific Ocean? Which one fashion at the ends of the branches. For this of the North American species is the most reason, the Rytidospermum magnolias are closely related to its Asian counterpart(s)? often referred to as umbrella . Indeed, to the Using modern molecular systematics, uninitiated, the first impression of these plants researchers Yin-Long Qiu, Clifford Parks, and is often more reminiscent of the houseplant Mark Chase analyzed the chloroplast DNA known as the umbrella tree-the giant tropical (cpDNA) of all section Rytidospermum species. Schefflera-than it is of a Magnoha. However, (CpDNA is the part of the DNA chromosome unlike Schefflera, whose compound leaves that is reponsible for photosynthesis. ~ By com- 25

paring the differences in the cpDNA of the (between all combmations of pairs of species) various species, they were able to quantify the over the entire chloroplast genome. The analy- amount of evolutionary change, measured as sis counts the number of site changes encoun- molecular divergence, that had taken place tered, then calculates the cpDNA sequence between them. The underlying assumption or divergence (as a percentage of sequence diver- theory in this method is that the amount of gence/ between all species pairs. The results are genetic difference is proportional to the amount shown in Table 1. of time elapsed since the species diverged from This analysis clearly shows that Magnolia their common ancestor, relative to other pairs tripetala from eastern North America has or groups of organisms being compared. The diverged far less from the Asian species M. results of the study team were published in two obovata and M. officinalis var. biloba than it separate papers in the American Journal of has from other North American species. It also Botany, both in 1995. This article attempts to indicates that the other North American species summarize the findings of these researchers and have diverged just as much from each other to interpret how molecular data, when used in (including M. tripetala) as they have from the conjunction with traditional morphological two Asian species. studies, can lead to better understanding of the evolutionary relationships among plants. No Allozyme Electrophoresis attempt will be made in this article to decode The study team used a second method, allo- the complexities of their analytic techniques, zyme electrophoresis, to examine genetic varia- the details of which are treated in the study tion of enzyme-coding genes. This analysis team’s original papers.4-s results in the calculation of a parameter called Nei’s unbiased genetic identity for each of the Restriction Site CpDNA Analysis species pairings. The numbers are from zero to Qui, Parks, and Chase used three different one, with one being a perfect genetic match. laboratory techniques to assess the divergence One of the authors, Clifford Parks, suggests that among Magnolia obovata, M. tripetala, M. as a rule of thumb, readings greater than 0.90 fraseri, M. macrophylla, and M. officmalis suggest populations of the same species, while var. biloba (a variety of M. officmalis with readings less than 0.67 indicate distinctly differ- notched or bilobed leaves; shown in the tables ent species. The results can be seen in Table 2. as M. biloba). The first method, cpDNA restric- Though not shown in the table, it should also be tion site analysis, randomly samples changes noted that Nei’s genetic identity for mtraspe-

TABLE 1 TABLE 2 26

cific comparisons was nearly 1.000, as would extremely low-the lowest divergence ever be expected: the values ranged from 0.993 for reported for any eastern Asia-eastern North M. obovata vs. M. obovata to 0.932 for M. America disjunct taxa." For example, the macrophylla vs. M. macrophylla. sequence divergence over the entire chloro- The results of this second method almost plast genome (cpDNA) between Limodendron mirror the results of the restriction site analysis, tulipifera and L. chinense was found to be 1.24 giving very strong evidence of a close relation- percent (as compared to 0.083 percent between ship between Magnolia tripetala and the Asian M. obovata and M. tripetala/,3 which is a species and relatively distant relationships remarkable difference in that many taxonomists among the rest of the species. It is interesting long considered both Liriodendron taxa to be to note that in both analyses the relationship varieties of the same species. between M. fraseri and M. macrophylla is The study team speculated how and when the most distant of any of the pairs. Iromcally, Magnolia tripetala and its sister species became some texts on North American trees refer separated from their common ancestor. One to these two species as closely related on hypothesis is that the common ancestor could account of their similar auriculate (earlobe- have migrated between the contments via the shaped) leaf bases. Bering land bridge during one of the earth’s warm periods in the middle Miocene (17 to 15 Gene rbcL Chloroplast Sequencing million years before the present) or early Finally, the study team comparedll Pliocene (6 to 5 million years before the tripetala, M. macrophylla, and M. TABLE 3 obovata to each other by analyzing (i.e., sequencing) a specific segment of the chloroplast gene called rbcL. This analysis involves comparing the 1,432 base pairs of the rbcL gene for each pair of species in the analysis, which in this case is three (M. macrophylla vs. M. obovata, M. macrophylla vs. M. tripetala, and M. obovata vs. M. tripetala). The results, once again, confirm the find- ings of the first two analyses, which suggest that M. tripetala and the two Asian species form a clade, or "sister group." In fact, the sequenc- ing of the chloroplast gene rbcL yielded no divergence between M. tripetala and M. obovata for that portion of the DNA strand. The researchers believe that since the results from all three methods have yielded the same pattern of divergence, they can be considered reliable for determining divergence among those Magnoha species. They that "the molecular emphasize The shared earlobe-shaped bases of their leaves notmthstandmg, divergence between M. tripetala and molecular analysis has shown that and, its Asian sister taxa, M. officin- seen here, M. frasen are the most distantly related of the alis var. biloba and M. obovata, is magnohas of section Rytidospermum. 27

Molecular analysis of Magnolia macrophylla var macrophylla (above) and M. macrophylla var ashen revealed no (below) differences between the two vametzes, despite the greater size of the vanety macrophylla, which attains sixty feet, as compared to that of vanety ashen, at twenty-five feet 28

among M. tripetala and the Asian M. of ficmalis var. biloba and M. obovata to include M. officmalis var. officinalis and M. rostrata in the group. Summaries of other findings follow: (1) Though Magnolia of ficm- alis var. officinalis and M. officinalis var. biloba are closely related and part of a sis- ter group, they are separated by four restriction site changes, whereas only one restriction site change separated M. officinalis var. officmalis and M. rostrata. This suggested to the team that "full species sta- tus for M. officinalis var. biloba is justifiable," but since delin- eation of a species depends on examination of samples from across a plant’s entire range, they recommend a detailed study of wild populations of all four Asian taxa before any deci- sion is made. (2) Only one restriction site change separated Magnolia macrophylla var. dealbata and M. macrophylla var. ashei, and no change was found between these two and M. macrophylla var. macrophylla. In this, the Above are thc .«. ~ , , o~ Magnolia frasem raz fiasem, and belov~, those of study team agrees with the M. frasen var pyramidata. Molecular analysis of these taxa revealed very 1979 judgment of botanist little separation between them Dorothy Johnson Callaway’ present). Perhaps the answer will come from and rejects species status for the varieties fossil evidence.3 dealbata and ashel. _ _ (3) Similarly, the team rejects species status the of the Expanding Scope Study for var. pyramidata since they In a second paper, Qui, Chase, and Parks found only one restriction site change between expanded their phylogenetic study to include it and M. fraseri var. fraseri. Also, separate restriction site analyses of many pairs of allozyme profiles established in an earlier study magnoliaceous species, including Magnolia of wild populations of M. fraseri at low eleva- officinalis var. officinalis, M. rostrata, M. tions in north Georgia indicated that those macrophylla var. ashei, M. macrophylla var. plants were intermediate between the varieties dealbata, M. fraseri var. dealbata, M. fraseri var. fraseri and pyramidata.6 pyramidata, as well as many others. One result For some, the ma~or finding of this work-the was an extension of the sister relationship sister relationship between Magnolia tripetala 29

and the Asian species (especially M. obovata), that result from genetic affinity between species comes as no surprise, smce these three are the from those that merely reflect similar responses only species that share grooved coats to similar environmental variables, such as cli- (the name Rytidospermum means "wrinkled mate. But lest false hope be raised, be warned seed") and are highly compatible when cross- that molecular analysis will not resolve the pollinated. Phil Savage, an experienced magno- arguments among taxonomists, nor, certainly, lia breeder, has found that of the many crosses does it offer respite from the frequent name he has made between species within section changes that have become such a predictable Rytidospermum, only those within the sister part of botanical . group were vigorous, worthy hybrids. In fact, where M. tripetala grows in close proximity to Literature Cited M. cases of obovata, there have been many 1 Johnson [Callaway], D. L. 1989. Nomenclatural putative hybrids occurring spontaneously.8-9 changes in Magnolia Baileya 23~1]: 55-56. Other crosses made x by Savage-M. tripetala 2 Kikuzawa, L 1989. Development and survival of M. M. obovata x M. fraseri, macrophylla, leaves in Magnoha obovata in a deciduous M. tripetala x M. macrophylla, and M. broadleaved forest in Hokkaido, northern Japan. obovata x M. fraseri-generally produced Canadian Journal of Botany 65(2) 412-417. smaller leaves and flowers than their parents, 3 Parks, C. R., and J. F. Wendel 1990. Molecular and all lacked vigor. divergence between Asian and North American Other affinities have been addressed using dif- species of Lmodendron (Magnohaceae/ with for of fossil floras. ferent morphological characters. Savage specu- implications interpretation American Journal of Botany 77: 1243-1256. lated that Magnolia obovata and M. fraseri may be closely related because of their long-beaked, ° Qm, Y.-L., M. W. Chase, and C. R. Parks. 1995 A carpelled , which are nearly identical.’ chloroplast DNA phylogenetic study of the eastern Some have with that of Asia-eastern North America disjunct section agreed point view, Ryudospermum of Magnoha (]. but others have argued that because all American Journal of Botany 82: 1582-1588. share a Rytidospermum magnolias very striking 5 morphological feature-the false whorls of Qm, Y.-L., C. R. Parks, M. W. Chase. 1995. Molecular divergence in the eastern Asia-eastern North leaves at the branch all produced tips-they America disjunct section Rytidospermum of must be closely related. However, since molecu- Magnoha (Magnoliaceae) American Journal of lar analysis suggests a close relationship for only Botany 82: 1589-1598 and its Asian sister Magnoha tripetala species, ~ Qm, Y.-L., and C. R. Parks. 1994. Disparity of perhaps the responsibility for the similar false allozyme variation levels in three Magnoha whorls produced by M. fraseri and M. macro- (Magnoliaceae) species from the southeastern Umted States. Amemcan phylla as well as the similarities in the fruit of Journal of Botany 81. 1300-1308 7 M. fraseri and M. obovata lies in convergent Savage, P. J. 1976. Sights and scents among the hardy evolution-that is, similar characteristics umbrella trees. Journal of the Magnoha Society may have developed in unrelated, or distantly 7/ 1 14-17. related, plants as each responds to similar 8 Spongberg, S. A., and R W. Weaver 1981 ’Silver conditions. Parasol’: A new magnolia cultivar. Arnoldia 41~2/: Molecular analysis as a taxonomic tool is still 70-77. 9 relatively new, and it brings with it the allure of Vasak, V. 1973 Magnoha hypoleuca m nature and results that can be stated in precise numbers. in cultivation. Journal of the Magnolia Society But taxonomy is far from a cut-and-dried proce- 9~ 1 3-6. dure : no matter how many characters are exam- ined and how much evidence is marshalled in Richard Figlar, a past president of the Magnolia support of a particular position, a taxonomic Society, has been studying and collecting magnolias for 25 decision is a call. As the case always judgment years. He grows more than 125 taxa in his personal with Magnolia demonstrates, molecular analy- arboretum in the foothills of the Blue Ridge Mountams sis does promise to help distinguish similarities of South Carolina.