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2Department of Biology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea; 3Department of Biology, Hallym University, Chunchon 200-702, Korea; and 4Natural Products Research Institute, Seoul National University, Seoul 110-460, Korea
Received for publication February 15, 2000. Accepted for publication June 20, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Magnoliaceae • molecular phylogeny • ndhF • sequences
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Mathews and Donoghue, 1999
; Parkinson, Adams, and Palmer, 1999
; Qiu et al., 1999
; Soltis, Soltis, and Chase, 1999
; Graham and Olmstead, 2000
). A fossil record also shows that the family has a long evolutionary history of over 100 million years (Dilcher and Crane, 1984
). The family is a well-defined group of trees and shrubs with over 230 species characterized by an androecium of numerous spirally arranged stamens, a gynoecium with many simple carpels spirally arranged on an elongated axis and separate tepals. All species of the family have bisexual flowers except for Kmeria and some species of Magnolia section Gynopodium (Chen and Nooteboom, 1993
). Four-fifths of the species are currently distributed in temperate and tropical regions of Southeast Asia, and the remaining one-fifth is found in America, from temperate southeast North America through tropical America to Brazil (Dandy, 1971
; Thorne, 1993
; Frodin and Govaerts, 1996
). The distribution of Magnoliaceae in eastern Asia and America is an outstanding example of intercontinental disjunction (Li, 1952, 1972
). The heterogeneous pattern of molecular divergence between several Asian and North American Magnolia species pairs suggests that the current distribution of Magnolia was attained by multiple migrations via both Bering and North Atlantic land bridges (Qiu, Parks, and Chase, 1995
).
Since Dandy (1927)
proposed the first comprehensive taxonomic treatment of the Magnoliaceae, many different infrafamilial taxonomic schemes have been suggested by various authors (Dandy, 1978
; Law, 1984
; Nooteboom, 1985, 1993
; Chen and Nooteboom, 1993
; Law, 1996
). Taxonomic treatment in the family has been controversial regarding the disposition of tribes, genera, and sections (Fig. 1). Dandy (1927, 1978)
recognized ten genera disposed in two tribes: Liriodendron in the tribe Liriodendreae, and Magnolia, Talauma, Aromadendron, Kmeria, Alcimandra, Manglietia, Pachylarnax, Elmerrillia, and Michelia in the tribe Magnolieae. Hutchinson (1959)
accepted two additional Chinese genera, Paramichelia H. H. Hu (1940)
and Tsoongiodendron W. Y. Chun (1963)
, in the tribe Magnolieae.
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proposed a view slightly different from the previous taxonomic treatments (Dandy, 1927, 1978
; Hutchinson, 1959
). He divided the family Magnoliaceae into two subfamilies, Magnolioideae and Liriodendroideae. Two tribes, four subtribes, and 14 genera were recognized in the former and the sole genus Liriodendron in the latter (Law, 1984
; Fig. 1). He accepted the genus Parakmeria, which was established from a Chinese species by Hu and Cheng (1951)
, including species of Magnolia section Gynopodium in Parakmeria. He also recognized Manglietiastrum as a distinctive genus (Law, 1979, 1984
).
In his comprehensive study of Magnoliaceae, Nooteboom (1985, 1987, 1993, 1998)
agreed with Law (1984)
on dividing the Magnoliaceae into two subfamilies, Magnolioideae and Liriodendroideae. He also recognized a sole genus Liriodendron in the subfamily Liriodendroideae, but subdivided the subfamily Magnolioideae into two tribes, Magnolieae and Michelieae (Nooteboom, 1985
). Thus the tribe Magnolieae consisted of four genera, Magnolia, Manglietia, Pachylarnax, and Kmeria, and the tribe Michelieae contained two genera, Elmerrillia and Michelia. In the genus Magnolia, he recognized three subgenera and 16 sections (Nooteboom, 1985
). Talauma, Dugandiodendron, Aromadendron, Alcimandra, Manglietiastrum, Tsoongiodendron, and Paramichelia, which were recognized as separate genera by previous authors (Dandy, 1927, 1978
; Hutchinson, 1959
; Law, 1984
), were merged as sections into the genus Magnolia and Michelia (Nooteboom, 1985
).
In their revision of Chinese Magnoliaceae, Chen and Nooteboom (1993)
adopted the classification system of Nooteboom (1985)
in broad outline. However, the section Manglietiastrum was transferred from the genus Magnolia to the genus Manglietia. In the treatment of Chinese Magnoliaceae, Law (1996)
proposed a very different classification system of Magnoliaceae from previous works (Dandy, 1927, 1950, 1978
; Hutchinson, 1959
; Law, 1984
; Nooteboom, 1985
; Chen and Nooteboom, 1993
). The most unusual feature of his classification was the inclusion of Illiciaceae and Schisandraceae in Magnoliaceae. These have generally been recognized as distinct families (Fig. 1). The inclusion of these families failed to gain general consent and is rejected by recent molecular phylogenetic analyses (Soltis et al., 1998
; Soltis, Soltis, and Chase, 1999
). Law (1996)
recognized the section Alcimandra as a separate genus in the subtribe Alcimandriinae of the tribe Magnolieae and included the genus Liriodendron in the tribe Michelieae (Fig. 1).
The controversies surrounding the taxonomy of Magnoliaceae are due to a paucity of phylogenetically useful characters caused by the extensive homogeneity in the family (Qiu, Chase, and Parks, 1995
; Nooteboom, 1998
). The variation of ndhF gene is second only to matK among coding genes in chloroplast DNA longer than 1000 bp (31% between rice and tobacco) (Olmstead and Palmer, 1994
). The ndhF gene has therefore been frequently used for phylogenetic studies at infrafamilial level (Olmstead and Palmer, 1994
; Clark, Zhang, and Wendel, 1995
; Kim and Jansen, 1995
; Olmstead and Reeves, 1995
; Scotland et al., 1995
; Bohs and Olmstead, 1997
; Oxelman, Backlund, and Bremer, 1999
). Comparative analysis of ndhF sequences of rice and tobacco demonstrates that the nucleotide substitution rate of ndhF is about two times higher than that of rbcL (Olmstead and Reeves, 1995
).
We examined the ndhF sequences to address phylogenetic questions in Magnoliaceae. The purpose of this study is to provide a well-supported phylogeny of Magnoliaceae capable of resolving controversies on infrafamilial groupings proposed by previous authors (Dandy, 1927, 1978
; Law, 1984, 1996
; Nooteboom, 1985
; Chen and Nooteboom, 1993
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Table 1), which we follow here. For the purpose of specific recognition, we adopted the scientific names listed in the recent bibliographic checklist of the Magnoliaceae by Frodin and Govaerts (1996)
.
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) or using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Total DNA extracted by CTAB was further purified with Geneclean Kit II (BIO101, Carlsbad, California, USA) for polymerase chain reaction (PCR). For species not available from other sources, extracted total DNAs were obtained from the DNA bank of the Royal Botanical Gardens Kew (Table 1).
For DNAs extracted from fresh materials, the entire ndhF gene was amplified using the primer pair of primer 1 developed by Olmstead and Sweere (1994)
and primer 14 by Jansen (1992)
(Fig. 2). For more degraded DNAs extracted from herbarium specimens, the ndhF gene was amplified in overlapping segments using the following pairs of primers: primer 1 by Olmstead and Sweere (1994)
and MF1165R designed by S. Kim, primer 972 and primer 2110R developed by Olmstead and Sweere (1994)
, and MF1795 designed by S. Kim and primer 14 developed by Jansen (1992)
(Fig. 2). Since the 3' primer, primer 14 developed by Jansen (1992)
, failed to work for certain taxa, the primer ORF-R (designed by S. Kim) was used for the amplification. Polymerase chain reaction was carried out in 100 µL final volume containing 0.5 ng template DNA, 2.5 units of Gold Taq polymerase (PE Applied Biosystems, Foster City, California, USA), 10 mmol/L Tris, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.001% gelatin, 200 µmol/L for each dNTP, and 0.5 µmol/L of each primer. Amplification reactions involved 10 min at 95°C for predenaturation, 30 cycles consisting of 1 min at 95°C for denaturation, 1 min at 55°C for annealing, and 3 min at 72°C for extension, with a final extension of 7 min at 72°C, using a Thermal Cycler 9600 (PE Applied Biosystems). The reaction was kept at 4°C after amplification.
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) either manually, or automatically in cycle sequencing reactions. For manual sequencing, double-stranded PCR products were directly sequenced using Sequenase PCR Product Sequencing Kit (USB, Cleveland, Ohio, USA). Electrophoresis was performed with denaturing formamide gel and glycerol tolerant buffer according to protocols for Sequenase PCR Product Sequencing Kit (USB). For automated sequencing, double-stranded PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN). The cycle sequencing reaction was carried out using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). In addition to PCR primers, the primers MF298, MF561, MF1254, MF1795, MF1945, MF256R, MF972R, MF1165R, and MF1861R (designed by S. Kim) were used as internal primers to complete sequencing in both directions (Fig. 2). Automated sequencing was employed with 377 DNA Sequencing System (PE Applied Biosystems).
Phylogenetic analysis
DNA sequences obtained from the automated DNA sequencer were assembled and consensus sequences were generated using the computer program Sequencher (version 3.1; Gene Codes Corporation, Ann Arbor, Michigan, USA). Sequences were aligned using CLUSTAL X (Thompson et al., 1997
). Phylogenetic analyses were performed by maximum parsimony (MP) and neighbor joining (NJ) using PAUP* (4.0 beta version; Swofford, 1998
). Trees were rooted by defining Liriodendron as the sister group to all other species of Magnoliaceae. Liriodendron is clearly distinguished from all other genera of Magnoliaceae based on both morphology and molecular data (Dandy, 1927, 1950, 1978
; Nooteboom, 1985
; Chase et al., 1993
; Chen and Nooteboom, 1993
; Qiu et al., 1993
). Searches for the MP tree, bootstrapping analysis, and decay analysis (Bremer, 1988
) were conducted using a standard heuristic search with MULPARS and Tree-Bisection-Reconnection (TBR) branch swapping. All nucleotide changes were equally weighted in the MP analysis (Terry, Brown, and Olmstead, 1997
). Searches for islands of MP trees (Maddison, 1991
) were conducted on 1000 replicates of random order entry addition of taxa, saving all optimal trees. Five hundred replicates were performed with the option of Maxtrees = 5000 to obtain bootstrap percentage. Batch command files for decay analysis were generated using AutoDecay (version 4.0; Eriksson, 1998
). The NJ tree was obtained by the distances calculated using Kimura's two-parameter method (Kimura, 1980
). Rates for variable sites were assumed to be equal. Minimum evolution criterion was chosen as the objective function, and negative branch-lengths were treated as zero length. In the bootstrap analysis of NJ trees, 1000 replicates were performed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) in species of Magnoliaceae examined, except for Magnolia macrophylla subsp. macrophylla, M. macrophylla subsp. ashei, M. dealbata, and Liriodendron chinensis. The length of the ndhF gene of these four taxa was three bases shorter at the 3' end of the gene. The difference in size was caused by nucleotide changes resulting in TGA (a stop codon) substituting for AGA (arginine) at 3' end (Fig. 3). Of 2199 sites, excluding the first 27 bases for the 5' PCR primer, 204 sites (9.3%) were variable and 124 sites (5.6%) phylogenetically informative. Variable sites in the ndhF gene were more densely distributed in the 3' end than at the 5' portion as noted in previous studies (Olmstead and Sweere, 1994
; Clark, Zhang, and Wendel, 1995
; Kim and Jansen, 1995
; Olmstead and Reeves, 1995
; Fig. 4). The G + C content of ndhF in the family Magnoliaceae was 34.4–35.0%. The maximum sequence divergence of the ndhF gene was 2.45% in the family Magnoliaceae (Kimura's K x 100; Kimura, 1980
) and 1.05 and 0.73% in the subfamily Magnolioideae and the subfamily Liriodendroideae, respectively. The sequence divergence in the Magnoliaceae was considerably lower than in other angiosperm families (Olmstead and Palmer, 1994
; Clark, Zhang, and Wendel, 1995
; Kim and Jansen, 1995
; Olmstead and Reeves, 1995
; Scotland et al., 1995
; Bohs and Olmstead, 1997
; Oxelman, Backlund, and Bremer, 1999
). The ratio of transition vs. transversion was 1.34, inferred from the MP analysis.
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Magnolia macrophylla subsp. macrophylla, M. macrophylla subsp. ashei, and M. dealbata, North American species of the section Rytidospermum, constitute a robust clade (Clade A) supported by a bootstrap value of 100%. It is sister to the rest of the subfamily Magnolioideae, but this position is weakly supported with a bootstrap value of just 39% (Fig. 5). In the remaining Magnolioideae (Clade B), seven distinctive clades (Clades I–VII) were recognized, but their relationships were poorly resolved due to the lack of synapomorphic changes (Fig. 5). The first clade constitutes the tribe Michelieae, section Maingola of Magnolia subgenus Magnolia, Magnolia subgenus Yulania, section Alcimandra of Magnolia subgenus Magnolia, section Aromadendron of Magnolia subgenus Talauma, Pachylarnax, section Manglietiastrum of Magnolia subgenus Talauma, and section Gynopodium of Magnolia subgenus Magnolia (Fig. 5, Clade I). This clade is divided into three subclades (Fig. 5, Clade Ia–c).
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Members of Magnolia subgenus Yulania form a well-defined group (Fig. 5, Clade Ib). Magnolia acuminata, the sole North American species of Magnolia subgenus Yulania represented here by two varieties, is placed at the base of the Yulania clade being separated from all other Asian species of the subgenus Yulania. In the Yulania clade, four species of section Yulania of Magnolia subgenus Yulania, M. dawsoniana, M. sargentiana, M. campbellii, and M. sprengeri, form a very distinctive clade separated by six synapomorphic changes with 100% bootstrap value.
Sections Gynopodium and Manglietiastrum of the genus Magnolia constitute a well-supported clade sharing five synapomorphic changes supported by 99% bootstrap value. The genus Pachylarnax is also included in the Gynopodium–Manglietiastrum clade (so Gynopodium is paraphyletic) and the ndhF sequences of P. praecalva and M. sinica were identical (Fig. 5, Clade Ic).
The genus Manglietia is strongly supported as monophyletic (Fig. 5, Clade II). All members of the section Theorhodon of Magnolia subgenus Magnolia, except for M. portoricensis and M. splendens, which are Caribbean species of section Splendentes sensu Vázquez-Garcia (1994)
, form a clade with M. virginiana, which is the sole member of the section Magnolia of Magnolia subgenus Magnolia (Fig. 5, Clade III). The section Gwillimia of Magnolia subgenus Magnolia is closely allied to section Lirianthe of Magnolia subgenus Magnolia, section Blumiana of Magnolia subgenus Talauma (Fig. 5, Clade IV), although they were considered to belong to different subgenera of Magnolia (Nooteboom, 1985
; Chen and Nooteboom, 1993
). The section Oyama of Magnolia subgenus Magnolia constitutes a moderately well-supported clade with Asian species of section Rytidospermum and M. tripetala, a North American species of the section (Fig. 5, Clade V).
New World subgenus Talauma constitutes a clade together with M. splendens and M. portoricensis. The latter two species are separated from the other species of section Theorhodon (Fig. 5, Clade VI). They were previously treated as section Theorhodon of Magnolia subgenus Magnolia, but Vázquez-Garcia (1994)
described them as a new section Splendentes. Magnolia fraseri, another North American species of section Rytidospermum, forms a poorly supported clade with Kmeria distributed in Southeast Asia (Fig. 5, Clade VII). As a result, the section Rytidospermum of Magnolia subgenus Magnolia is divided into three independent lineages, implying that the traditionally recognized section Rytidospermum is polyphyletic (Fig. 5, Clades A, V, and VII).
The NJ tree presents a basically identical topology among major clades with the MP tree in spite of very different assumptions to construct trees (Fig. 6). Both the MP and NJ trees recognized nine major independent lineages including Liriodendron in the Magnoliaceae (Figs. 5, 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Clark, Zhang, and Wendel, 1995
; Kim and Jansen, 1995
; Olmstead and Reeves, 1995
; Scotland et al., 1995
; Bohs and Olmstead, 1997
; Oxelman, Backlund, and Bremer, 1999
). The low sequence divergence and the long evolutionary history of the family Magnoliaceae in the fossil record (Dilcher and Crane, 1984
) indicate that the ndhF gene has evolved slowly in the family. It frequently has been claimed that molecules evolve more slowly in perennial woody plants than in annual herbaceous plants (Wilson, Gaut, and Clegg, 1990
; Bousquet et al., 1992
; Chase et al., 1993
; Suh et al., 1993
). The ndhF gene in the Magnoliaceae gives an excellent example of the retarded evolutionary rate in woody perennial plants.
Recognition of subfamilies Magnolioideae and Liriodendroideae
It is generally accepted that Magnoliaceae should be divided into two subfamilies, Magnolioideae and Liriodendroideae (Law, 1984
; Chen and Nooteboom, 1993
; Nooteboom, 1998
). The latter, with the sole genus Liriodendron of two species, is clearly distinguished from the former by easily recognizable features, such as 2–10 lobed leaves, extrorsely dehiscing anthers, and winged, deciduous and indehiscent samaroid fruits. Molecular phylogenetic analyses based on rbcL sequences also strongly support the separation of two subfamilies in the Magnoliaceae (Chase et al., 1993
; Qiu et al., 1993
). For these reasons, trees were rooted to separate the subfamily Liriodendroideae from the subfamily Magnolioideae in the ndhF analyses. Moreover, since the sequence divergence value of the ndhF between the subfamily Liriodendroieae and the subfamily Magnolioideae (2.45%) are higher than those within Magnolioideae (1.05%) and Liriodendroideae (0.73%), respectively, the division into two subfamilies seems secure.
Michelieae–Yulania–Gynopodium aggregate
It has been generally agreed that subfamily Magnolioideae should be subdivided into two tribes, Magnolieae and Michelieae (Law, 1984
; Nooteboom, 1985
). Axillary flowers distinguish tribe Michelieae from tribe Magnolieae, which has terminal flowers. However, the ndhF molecular tree does not support the separation based on the flower position (Figs. 5, 6). Although flowers of Michelia and Elmerrillia (tribe Michelieae) have often been perceived to be axillary, flower buds are actually produced terminally on short shoots (brachyblasts) arising from the leaf axis (Nooteboom, 1985
; Figlar, 2000
).
In the ndhF tree, species in section Maingola of Magnolia subgenus Magnolia, M. griffithii, M. pealiana, and M. gustavii, are nested in Michelia, and then the clade of the tribe Michelieae is closely related to the clade of Magnolia subgenus Yulania. The close alliance of Maingola with Michelia has never been proposed, but species of Maingola, Michelia, and Yulania have cylindrical fruits in common (Dandy, 1978
). One of the main features separating Magnolia subgenus Yulania from subgenus Magnolia relates to the dehiscence of anthers. Pollen is shed introrsely in subgenus Magnolia, but laterally in the subgenus Yulania. In Michelia, pollen is also shed laterally (Dandy, 1978
). The close relationship between Michelia and Yulania is also demonstrated by proleptic growth and the formation of hybrids (Figlar, 2000
). Michelia and the subgenus Yulania have been observed to form branches by prolepsis, in which branches are produced from a dormant axillary bud of the previous year's growth. On the other hand, all species of Magnolia subgenus Magnolia, except for section Oyama, produce branches directly from the current year's growth by syllepsis (Tomlinson, 1983
; Figlar, 2000
). In addition, the close affinity between Michelia and Yulania is demonstrated by their cross compatibility. A hybrid was successfully produced between Michelia figo and M. acuminata, a species of subgenus Yulania, but many attempts to produce hybrids between the two subgenera Magnolia and Yulania have never been successful (Savage, 1989
; Figlar, 2000
).
Magnolia cathcartii, the sole member of section Alcimandra of Magnolia subgenus Magnolia, was originally described as a species of Michelia (Hooker and Thomson, 1855
), and Lozano-Contreras (1975)
recognized that this species has pseudolateral flowers. Later, Dandy (1927)
separated this taxon as the independent genus Alcimandra because other species of Michelia comprise a well-defined group with consistently axillary flowers. However, since the axillary flowers are interpreted to be actually terminal on brachyblasts, the current taxonomic position of M. cathcartii should be reconsidered. In fact, Alcimandra has been treated as a distinctive genus from Magnolia because it has stipitate gynoecium similar to the condition in all species of Michelia (Nooteboom, 1985
; Figlar, 2000
). The gynophore develops even further in fruit to a short stalk on the fruiting axis between the androecium and the base of gynoecium.
A stipitate gynoecium is also found in Pachylarnax, section Manglietiastrum of Magnolia subgenus Talauma, and section Gynopodium of Magnolia subgenus Magnolia, which together form Clade Ic. Section Manglietiastrum is similar to the genus Manglietia but distinguished by petioles without stipular scars and a gynoecium with a short gynophore (Chen and Nooteboom, 1993
). In the ndhF tree, Manglietiastrum is quite distantly related to the clade of Manglietia. Manglietiastrum is separated from the well-defined clade of Manglietia and allied with P. praecalva and species of section Gynopodium of Magnolia subgenus Magnolia. Although Manglietia was once considered to belong to the genus Magnolia (Baillon, 1866
), it has been recognized as a distinctive genus defined by four or more ovules in each carpel (Law, 1984
; Nooteboom, 1985
). Manglietiastrum often has been classified as an independent genus (Law, 1979, 1984, 1996
) and sometimes as a section of Magnolia subgenus Talauma (Nooteboom, 1985
) or a section of Manglietia (Chen and Nooteboom, 1993
). The ndhF tree does not support a close association of Manglietiastrum with Manglietia or with other species of Talauma.
Gynopodium has been considered to be a section of Magnolia subgenus Magnolia (Dandy, 1978
; Nooteboom, 1985
; Chen and Nooteboom, 1993
) or designated as the genus Parakmeria (Hu and Cheng, 1951
; Law, 1984, 1996
). The ndhF molecular tree strongly suggests that the clade of Pachylarnax–Manglietiastrum–Gynopodium is closely related to the Michelieae and Yulania clades. This association is also supported by the character of a stipitate gynoecium.
Pachylarnax has been considered to be a well-distinguished genus based on the unique capsular fruit (Dandy, 1978
; Law, 1984, 1996
; Nooteboom, 1985). Pachylarnax praecalva is placed together with species of sections Manglietiastrum and Gynopodium in the ndhF tree. The ndhF sequence of P. praecalva is identical with that of M. sinica, the only species of section Manglietiastrum, implying a strong association.
Michelia odora was once considered a monotypic genus, Tsoongiodendron, characterized by crowded, sessile, woody, and large fruits (Chun, 1963
). Michelia baillonii was also regarded as the distinctive genus Paramichelia because it has syncarpous fruits and entirely adnate stipules (Hu, 1940
). The separation of Toongiodendron and Paramichelia as distinctive genera is not supported by the ndhF analysis.
Magnolia subgenus Yulania
The subgenus Yulania has been divided into three sections, Tulipastrum, Yulania, and Buergeria (Dandy, 1927, 1950
). These sections are recognized by (1) the presence/absence of sepaloid tepals, the tepals of the outermost whorl being smaller than the inner tepals, (2) the time of flowering before or after the production of leaves, and (3) the color of the tepals (Dandy, 1927, 1950
). In the ndhF tree, M. acuminata, the sole North American species of the subgenus Yulania, is separated from the Asian species and placed at the base of the Yulania clade. Traditionally, M. liliiflora, which is an Asian species, and M. acuminata constitute section Tulipastrum because of the sepaloid tepals and flowers appearing with or after the production of leaves (Dandy, 1927, 1950
; Nooteboom, 1985
; Chen and Nooteboom, 1993
). However, molecular data show that M. liliiflora is quite distantly related to M. acuminata. The ndhF sequence of M. liliiflora is identical with those of M. denudata and M. cylindrica that belong to section Yulania and Buergeria, respectively. In addition, since other species of section Yulania are variously related to species of section Buergeria, the ndhF data do not support sectional treatments of the subgenus Yulania based on sepaloid tepals, time of flowering, and the tepal color (Chen and Nooteboom, 1993
). Four species of the section Yulania, M. dawsoniana, M. sargentiana, M. campbellii, and M. sprengeri, form a strongly supported clade, but there are no discrete characters that define this clade except for the relatively large flowers with pinkish tepals.
Clade of sections Theorhodon–Magnolia of Magnolia subgenus Magnolia
Recently Vázquez-Garcia (1994)
separated Caribbean species (M. portoricensis and M. splendens) from North and Central American taxa in section Theorhodon and placed them in section Splendentes. Section Splendentes is distinguished by stamens with the connective apex extended into a long setiform appendage, while species of Theorhodon have stamens with the short connective apex acute to acuminate. The stamen appendages of Splendentes become embedded in the gynoecium and support the stamen when it detaches at the base during dehiscence of the anther (Howard, 1948
; Vázquez-Garcia, 1994
). The ndhF data strongly support this segregation of section Splendentes by Vázquez-Garcia (1994)
. Splendentes is separated from the clade of Theorhodon and associated with South American Talauma, being closely allied with M. lenticellatum. Magnolia lenticellatum, a Colombian species of Talauma that was treated as section Dugandiodendron by Lozano-Contreras (1975)
, also has a long, elongate, hair-like appendage at the tip of stamen. Magnolia virginiana, which is a sole member of the section Magnolia of Magnolia subgenus Magnolia, is closely allied with the core members of Theorhodon in the ndhF tree. The close affinity between M. virginiana and section Theorhodon has also been demonstrated by chloroplast DNA restriction fragment length polymorphism (RFLP) comparison without the inclusion of Splendentes in the analysis (Qiu, Chase, and Parks, 1995
).
Clade of sections Gwillimia, Lirianthe, and Blumiana
Sections Gwillimia and Lirianthe of Magnolia subgenus Magnolia are placed together, forming a clade. Both sections are distinguished by beaked fruiting carpels, and the beak of the monotypic Lirianthe is longer than those found in species of Gwillimia and forms a dorsally flattened coriaceous appendage that becomes more or less curved (Nooteboom, 1985
). It should be noted that Southeast Asian members of Talauma, sections Blumiana and Aromadendron, are clearly separated from New World Talauma, placed in the clade of section Gwillimia. The close relationship between Blumiana and Gwillimia previously has been suggested because they are almost impossible to distinguish without fruits, although have been assigned to different subgenera (Nooteboom, 1985
; Chen and Nooteboom 1993
). Sections Aromadendron and Blumiana were assigned to subgenus Talauma because of connate carpels in fruits (Dandy, 1978
; Nooteboom, 1985
). The ndhF data suggest that subgenus Talauma, as traditionally recognized by connate carpels, is polyphyletic. Since Southeast Asian Talauma is distantly related to New World Talauma, and Splendentes of Magnolia subgenus Magnolia closely associates with New World Talauma, the taxonomic circumscription of subgenus Talauma should be adjusted.
Sections Rytidospermum and Oyama of Magnolia subgenus Magnolia
The most striking character that distinguishes section Rytidospermum is the whorl-like arrangement of the leaves, as indicated by the name of umbrella tree commonly used for the American species (Dandy, 1978
). Dandy (1978)
recognized three distinctive lineages in Rytidospermum: (1) Asian series comprising M. hypoleuca, M. officinalis, and M. rostrata, (2) American series of M. tripetala, M. fraseri, and M. pyramidata (= M. fraseri var. pyramidata), and (3) American series of M. macrophylla, M. ashei (= M. macrophylla subsp. ashei), and M. dealbata. The ndhF analysis clearly distinguishes three lineages, and the only discrepancy is in the placement of M. tripetala.
Magnolia tripetala forms a clade together with the Asian series, which has a close affinity with the section Oyama. All species of Oyama are also Asian. The close affinity between M. tripetala and Asian Rytidospermum is supported by the extremely similar seed and fruit morphology, identical rbcL sequences of M. tripetala and M. obovata (= M. hypoleuca), high genetic identity estimated from allozyme variation, high interspecific cross compatibility between M. tripetala and Asian species, and chloroplast DNA RFLP analysis (Parks et al., 1983
; Qiu et al., 1993
; Qiu and Parks, 1994
; Qiu, Chase, and Parks, 1995
; Qiu, Parks, and Chase, 1995
). Evidence that Rytidospermum was polyphyletic resulted from cladistic analysis of chloroplast DNA RFLP. Thus, leaf morphology and wood anatomy shared by Asian and North American Rytidospermum were assumed to be convergent (Qiu, Chase, and Parks, 1995
). The more extensive sampling of Magnoliaceae presented here supports that conclusion.
Magnolia fraseri constitutes a distinctive lineage, separated from the clade of Asian Rytidospermum that contains North American species, M. tripetala. Another group of North American Rytidospermum, M. macrophylla and M. dealbata, is placed at the base of the ndhF tree, suggesting they might be the sister group to all other species of the Magnolioideae. Since this position is poorly supported, additional data will be required to identify the earliest split in Magnoliaceae. The basal placement of M. macrophylla and M. dealbata was also shown in the chloroplast DNA RFLP analysis (Qiu, Chase, and Parks, 1995
). In the ndhF sequences, M. macrophylla subsp. macrophylla, M. macrophylla subsp. ashei, M. dealbata, and Liriodendron chinensis share a three-base deletion at the end of the gene. However, the significance of this deletion is unclear because the other outgroup species, L. tulipifera, does not have the deletion. In the phylogenetic analysis of rbcL sequences, M. macrophylla is also placed at the base among the clade of four species of Magnolia, which is closely associated with Liriodendron (Qiu et al., 1993
).
Phylogenetic position of Kmeria
Kmeria, distributed in Southeast Asia, has been treated as a distinctive genus because it has unisexual flowers (Dandy, 1927, 1978
; Law, 1984
; Nooteboom, 1985
; Chen and Nooteboom, 1993
) and its phylogenetic associations have never been proposed. The close association of Kmeria with M. fraseri in the ndhF tree remains in question because no other evidence so far supports the relationship. This affinity may rather be explained by long-branch attraction because the Kmeria–M. fraseri clade is supported by only one base change at the third position in codon, which is considerably less in comparison to four and nine steps supporting the clades of M. fraseri and Kmeria, respectively (Felsenstein, 1978
; Hendy and Penny, 1989
).
Conclusions
This study is the first attempt to elucidate phylogenetic relationships in the family Magnoliaceae from a comprehensive sampling of taxa representing all sections recognized to date. Phylogenetic analysis of Magnoliaceae using ndhF sequences not only confirmed many taxonomic relationships based on morphological data, but also provided evidence for phylogenetic relationships previously undetected by systematists. Although ndhF sequences do not completely resolve phylogenetic relationships in the family, they clearly delimit major lineages that are supported by other data. This study has also demonstrated that ndhF data are phylogenetically informative despite low sequence divergence within Magnoliaceae. Examination of rapidly changing genes and multiple gene analysis, part of an ongoing endeavor, will certainly enhance our understanding of the phylogeny of the family, which is essential to generate a natural classification system.
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5 Author for reprint requests.
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LITERATURE CITED |
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