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Systematics |
2Department of Higher Plant Systematics and Evolution, Institute of Botany and Botanical Garden, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; 3Jodrell Laboratory, Section of Molecular Systematics, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3DS UK; 4Daubeny Herbarium, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RA UK; 5Department of Comparative and Ecological Phytochemistry, Institute of Botany and Botanical Garden, University of Vienna, Rennweg 14, A-1030 Vienna, Austria
Received for publication April 30, 2004. Accepted for publication November 16, 2004.
ABSTRACT
We performed maximum parsimony and Bayesian analyses (nuclear ITS rDNA, plastid rps16 intron) to estimate phylogenetic relationships within Aglaia (over 100 species in Southeast Asia, the Pacific, and Australia) and its relations among Aglaieae (Meliaceae). Based on 67 accessions of Aglaieae, three taxa of Guareae, and two taxa of Melieae (outgroup), this study provides the first assessment of the current circumscription of Aglaieae, Aglaia, and its sections and to a more limited extent of species concepts in Aglaia. DNA data are compared to recently collected data on chemical profiles. Our analyses indicate (1) the monophyly of Aglaieae; (2) the polyphyly of Aphanamixis; (3) the paraphyly of Aglaia; (4) the existence of at least three entities with respect to Aglaia: (a) the core group of Aglaia section Amoora (dehiscent fruits) with close relationships to Lansium and Reinwardtiodendron, (b) a group comprising morphological intermediates between the two sections, and (c) the core group of Aglaia section Aglaia (indehiscent fruits). Macro- and micromolecular data indicate that complex species are more heterogeneous, i.e., probably containing more than one taxon each, than taxonomically isolated species. A third section in Aglaia is recognized to accommodate A. lawii, A. teysmanniana, and A. beccarii.
Key Words: Aglaia • chemotaxonomy • internal transcribed spacer (ITS) • Meliaceae • molecular phylogenetics • plastid ribosomal protein gene intron (rps16) • Sapindales
Aglaia Lour., the largest genus of the subtropical and tropical angiosperm family Meliaceae (order Sapindales), contains at least 106 (115) arborescent species and presents more taxonomic problems in species delimitation than any other genus of the family (Pannell, 1992
, 1998a
, b
; Mabberley et al., 1995
; Pannell, 2004
). Aglaia forms an important component of the moist tropical forest in the Indomalesian region. The total range of the genus comprises the tropics of Southeast Asia from Sri Lanka and India to Australia (Queensland, Northern Territory, and Western Australia) and as far east as the island of Samoa in Polynesia and north to the Marianne (Saipan, Roti, and Guam) and Caroline Islands (Palau and Ponape) in Micronesia (Pannell, 1992
).
During the past few years, the genus has received increasing scientific focus due to its bioactivity potential. Flavaglines, especially cyclopenta[b]benzofurans, were shown to be potent insecticides (Brader et al., 1998
; Bacher et al., 1999
; Nugroho et al., 1999
; Dreyer et al., 2001
; Greger et al., 2001
). In addition to that, cytotoxic (Cui et al., 1997
) and antifungal (Engelmeier et al., 2000
) effects were found for these compounds, so far only known from Aglaia species. Other classes of natural products occurring in Aglaia include lignans (Brader et al., 1998
; Greger, 2000
; Wang et al., 2001
), flavonoids, and bisamides (Greger et al., 2000
, 2001
), the last also only known from Aglaia; some of these exhibit cytotoxic and antiviral properties (Saifah et al., 1993
, 1999
). Furthermore, many terpenoids are reported (Fuzzati et al., 1996
; Brader et al., 1998
; Mohamad et al., 1999
; Puripattanavong et al., 2000
; Weber et al., 2000
; Greger et al., 2001
). However, so far no chemotaxonomic framework exists for Aglaia. In contrast, at higher taxonomic levels first attempts have been made to use limonoids (bitter tetranortriterpenoids) as marker compounds that might offer possibilities for assessing intergeneric taxonomy in Meliaceae (e.g., Taylor, 1981
; Mulholland et al., 1998
), but knowledge is still so fragmentary (Mabberley et al., 1995
) that comprehensive phylogenetic conclusions (Da Silva et al., 1984
, 1999
; Agostinho et al., 1994
; Neto et al., 1998
) have either been premature or conflicting.
Besides its unique biogenetic trend to produce biologically highly active flavaglines, Aglaia is morphologically distinguished from most other genera in Meliaceae by its characteristic indumentum of peltate scales or stellate hairs; simple hairs are never found on vegetative parts of the plant (Pannell, 1992
). Lepidotrichilia (Harms) J. F. Leroy, Astrotrichilia (Harms) J. F. Leroy, Pterorhachis Harms, and Chisocheton section Rhetinosperma (Radlk.) Mabberley also possess a stellate indumentum, and Trichilia rarely has stellate or peltate scales. These genera might therefore be confused with Aglaia on indumentum alone, but they are easily separated on floral characters. When stellate hairs or scales occur in genera other than these, their structure is different; they are usually either forked hairs or clumps of simple hairs [e.g., Melia, Aphanamixis polystachya (Wall.) R. N. Parker] and nearly always interspersed with simple hairs. The shoot apices in these taxa are densely covered with simple hairs, not stellate hairs or peltate scales (Pannell, 1992
). However, well-defined sets of morphological characters that can be used to define genera are clearly absent in Aglaieae, which is reflected by the complicated taxonomic history of Aglaia.
Tribe Aglaieae, including Aglaia, Aphanamixis Blume (three species), Lansium Correa (three species), Reinwardtiodendron Koord. (seven species), and Sphaerosacme Wall. ex Royle (one species), was first described by Blume (1825)
and owes its present circumscription to the work of Pennington and Styles (1975)
. In the past, Amoora had been separated from Aglaia because of differences in the number of anthers (de Candolle, 1878
) or confined to either those species with three petals (King, 1895
) or those with dehiscent fruits (Harms, 1940
). The inclusion of Aphanamixis and Sphaerosacme decandra (Wall.) Pennington in Amoora by some authors (e.g., de Candolle, 1878
) added to the confusion. Pellegrin (1911)
also considered that Lansium should be included in Aglaia, and Kostermans (1966)
combined the two genera. His Aglaia section Lansium contained 7–8 species of Lansium, six of Reinwardtiodendron, and one or two of Lepisanthes Blume (Sapindaceae; see Pennington and Styles, 1975
; Mabberley, 1985
). In the most recent account of Aglaia, Pannell (1992)
followed Pellegrin (1911)
and Pennington and Styles (1975)
, who reduced Amoora to Aglaia. Pennington and Styles (1975)
united Amoora and Aglaia because the sets of character state pairs previously used to separate them were not sufficiently correlated in all species, and there was a gradation in the structure of their secondary xylem. Pannell (1992)
recognized two sections in Aglaia, Aglaia and Amoora, separated by fruit dehiscence. Two species, Aglaia lawii and A. teysmanniana, have flower characters intermediate between the two sections, but they were placed in section Amoora because of their dehiscent fruits (Pannell, 1992
).
In her recent taxonomic treatment of the genus, Pannell (1992
, 1998a
, b
) adopted a wide species concept, and thus for many species even the most indicative morphological characters, such as indumentum, fruit, and floral morphology, vary considerably. Pannell (1992)
recognized different types of species in her monograph of the genus. "Isolated species" are morphologically distinct species without any close relatives and with either small (e.g., A. coriacea Korth. ex Miq.) or extensive (e.g., A. cucullata (Roxb.) Pellegrin) geographical distribution. In contrast, members of closely related pairs or larger groups of species are often separable only by using the combined variation of several overlapping characters. Members of these groups may be allopatric (e.g., A. elliptica Blume and A. cinnamomea Baker fil.; Pannell, 1993
) or sympatric (e.g., A. korthalsii Miq. and A. speciosa Blume). In "variable species," variation is relatively simple, usually involving two variants linked by intermediates. "Complex species" have a more extensive, complicated, and putatively reticulate pattern of variation, for which extremes appear at first sight to belong to distinct species (Pannell, 1992
, 1998a
, b
).
The controversial taxonomic history of Aglaieae and Aglaia and the lack of consensus about taxon delimitation at all taxonomic levels based on morphology make it clear that there is need for additional research. Molecular techniques can be used to investigate genetic diversity and relationships among species. These data provide helpful tools for taxon delimitation especially in plant groups in which the number of diagnostic morphological characters is limited and parallel evolutionary trends might obscure phylogenetic relations (Muellner et al., 2003
). Moreover, during the past few decades biodiversity within Aglaia, constituting one of the most important sources of biologically active compounds within Meliaceae, has become severely threatened due to habitat loss. Currently, 95 species of Aglaia are included in The World Conservation Union Red List (IUCN, 2003
). However, measures for conserving biodiversity are only effective if species are genetic entities, i.e., taxonomic species reflect evolutionarily distinct units. This emphasizes the importance of reevaluation of the taxonomic framework for Aglaia.
Phylogenetic studies have demonstrated that the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA), defined as the unit containing the ITS1 spacer, 5.8S rRNA gene, and ITS2 spacer, are useful in assessing relationships at infrageneric levels (e.g., Baldwin and Markos, 1998
; Whitten et al., 2000
; Vaasen et al., 2002
; Chase et al., 2003
). Plastid noncoding regions like the rps16 intron have also been shown to be suitable for inferring phylogenetic relationships at lower taxonomic levels (Edwards and Gadek, 2001
; Popp and Oxelman, 2001
; Wanntorp et al., 2001
; Clarkson et al., 2002
). Combined analyses of rps16 intron and ITS sequences have been shown to provide a useful tool for resolving difficult taxonomic issues (Oxelman et al., 1997
).
In this study, we performed maximum parsimony and Bayesian analyses of sequence data from these two regions (nuclear ITS and plastid rps16 intron) to estimate phylogenetic relationships within tribe Aglaieae and within its largest genus Aglaia. The aim of our investigation was to assess the current circumscription of (1) tribe Aglaieae, (2) Aglaia, and (3) sections, and (4) the wide species concept proposed for Aglaia by Pannell (1992)
. The DNA data are compared to recently collected data on chemical profiles of the respective taxa of Aglaia to investigate if phylogenetic relationships between species and within complex species are reflected by biogenetic trends.
MATERIALS AND METHODS
We analyzed ITS and rps16 intron data of 67 accessions of Aglaieae, including representatives and type species of both currently recognized sections within Aglaia (Amoora and Aglaia), three accessions of Guareae, and two accessions of Melieae (outgroup), all being members of Melioideae (Appendix, see Supplementary Data accompanying the online version of this article). Intrafamilial phylogenetic relationships of Meliaceae were previously assessed by an evaluation of the higher-level classification using DNA sequence data from three regions: plastid genes rbcL, matK, and nuclear 26S rDNA (Muellner et al., 2003
). Guareae are the tribe genetically closest to Aglaieae (Muellner et al., 2003
; A. N. Muellner, R. Samuel, M. W. Chase, T. F. Stuessy, unpublished manuscript). For most of the Aglaia species referred to as "variable" or "complex" (Pannell, 1992
), 2–5 individuals were included in this study.
Plant material
Plant material of ingroup taxa was collected during excursions to Bangladesh, Thailand, Fiji, Samoa, and Australia or taken from herbarium specimens. Outgroup taxa Azadirachta and Melia were collected during excursions to Sri Lanka and from the living collections of the Royal Botanic Gardens, Kew, UK. Herbarium specimens, including the newly collected material, are deposited at the University of Vienna Herbarium (WU), Austria, or the Herbarium of the Royal Botanic Gardens Kew (K), UK (Appendix, see Supplementary Data accompanying the online version of this article).
Isolation of DNA, amplification, and sequencing
Field-collected material was dried in silica gel prior to DNA extraction (Chase and Hills, 1991
). Total DNA was extracted from similar amounts of silica-gel-dried tissue as well as from herbarium specimens following the cetyltrimethyl-ammonium bromide (CTAB) procedure of Doyle and Doyle (1987)
with the following modifications: after precipitation with isopropanol and subsequent centrifugation, the DNA pellet was washed with 70% ethanol, dried at 37°C, and resuspended in TRIS-EDTA (TE) buffer. Polymerase chain reaction (PCR) amplification was carried out in a PTC-100 Programmable Thermal Controller (MJ Research, Margaritella, Bio-Trade, Vienna, Austria) using the following primers: 17SE and 26SE (Sun et al., 1994
), ITS4 (White et al., 1990
), as well as the newly created primer pair F1-ITS (5'-GATCGCGGCGACTTGGGCGGTTC-3') and R1-ITS (5'-GGTAGTCCCGCCTGACCTGGG-3') for the fragment comprising part of 18S rDNA, the entire ITS region and part of 26S rDNA; rpsF, rpsR2 (Oxelman et al., 1997
) as well as the newly created primer pair F-rps (5'-ATCCGCTATGGATTTCTTTACATC-3') and R-rps (5'-CTCTCATAACTCAAGTTGGATAAC-3') for the rps16 intron (partial). A 50-µL reaction mix contained 45 µL 1.1 x ReddyMix PCR Master Mix (2.5 mmol MgCl2; Advanced Bioenzymes, Surrey, UK), 1 µL of the primers each (20 pmol), 1 µL template DNA (100–2000 ng/µL), as well as 2 µL dimethyl sulfoxide (DMSO) for ITS and 5 µL bovine serum albumin (BSA; 0.4%) for the rps16 intron. These additives are thought to stabilize the enzyme (BSA), reduce secondary structure problems (DMSO; e.g., within-strand Watson-Crick pairing of ITS; Baldwin et al., 1995
), or favor precise annealing (DMSO; Palumbi, 1996
). Amplification and gel purification of amplification products were carried out according to Muellner et al. (2003)
, with slight modifications for some taxa. The same primers as described earlier were used for sequencing. Sequencing was carried out according to Muellner et al. (2003)
.
Sequence editing and alignment
For editing and assembly of the complementary strands, the software programs Autoassembler version 1.4.0 (Applied Biosystems) and DNA STRIDER version 1.2 (Christian Marck, CEA— Commissariat à L'ènergie Atomique/Saclay, France) were used. The ITS was explored for the presence of several structural motifs to check for the potential occurrence of pseudogenes. Thus, in the ITS1 region, we searched for the presence of the conserved angiosperm motif GGCRY—(4 to 7 n)—GYGYCAAGGAA (Liu and Schardl, 1994
). We also looked for the presence of the conserved (C1–C6) and variable (V1–V6) domains determined for plant ITS2 sequences (Hershkovitz and Zimmer, 1996
), as well as for the conserved angiosperm motif 5'-GAATTGCAGAATCC-3' within the 5.8S rDNA gene, which can be used to differentiate between flowering plants, fungi, and algae (Jobes and Thien, 1997
). Folding predictions of secondary structures of the ITS1 and ITS2 RNA transcripts were made at the M. Zuker web server (http://www.bioinfo.rpi.edu/
zukerm/) using the mfold program version 3.1 (Mathews et al., 1999
; Zuker et al., 1999
). Folding was conducted at 37°C. After a first rough alignment of sequences using CLUSTAL version X (Thompson et al., 1997
), corrections were made manually using secondary structure predictions of ITS1 and ITS2 RNA transcripts as a guide for alignment across genera. A total of 741 and 701 nucleotides were included in the matrices for phylogenetic analyses for ITS (including ITS1, 5.8S rDNA, and ITS2) and for rps16 (partial intron sequence), respectively. Gaps were coded as missing data. All sequences have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/). Aligned matrices are available from A. N. Muellner and M. W. Chase (a.muellner@kew.org; m.chase@kew.org).
Maximum parsimony (MP) analysis
Individual and combined maximum parsimony (MP) analyses of the ITS and rps16 intron data sets were performed using PAUP*4.0b10 (Swofford, 2002
). Visual inspection of the individual bootstrap consensus trees was used for determining congruence of the two data sets (Whitten et al., 2000
). Because there were no strongly supported (<85% bootstrap), incongruent patterns in the individual trees, direct combination was regarded as appropriate. Substitutions at each nucleotide position were treated as independent, unordered, multi-state characters of equal weight (Fitch parsimony; Fitch, 1971
). Heuristic searches were performed using addition sequence set at 1000 random additions of taxa, tree bisection-reconnection (TBR) branch swapping, and MulTrees on (keeping multiple, shortest trees) but holding only 10 trees per replicate to reduce time spent in swapping on large numbers of trees. After these 1000 replicates, we then used the shortest trees found as starting trees for a swapping-to-completion search (with a tree limit of 15 000 for the rps16 data set). Robustness of clades was estimated using the bootstrap (Felsenstein, 1985
) with 1000 replicates with simple sequence addition, TBR branch swapping, and MulTrees on but holding only 10 trees per replicate to reduce time spent on each replicate (Salamin et al., 2003
). We conducted a separate analysis of the rps16 intron, but the level of variation was so low that the strict consensus tree is highly unresolved, and because of its lack of informativeness we do not illustrate it here. Combining this matrix with that of ITS did produce greater resolution and bootstrap support (results not shown) so collecting these data was worthwhile in spite of their highly unresolved pattern when analyzed alone.
Bayesian analysis
A number of numerical methods are available that allow the posterior probability of a tree to be approximated, the most useful of which is Markov chain Monte Carlo (MCMC). We conducted Bayesian analysis in MrBayes version 2.01 (Huelsenbeck and Ronquist, 2001
) on the combined ITS/rps16 data matrix using four Markov chains simultaneously started from random trees. Modeltest 3.06 (Posada and Crandall, 1998
, 2001
) was used to select the optimal substitution model (general time reversible model, GTR). One million cycles were performed, sampling a tree at every 100 generations. Trees that preceded the stabilization of the same likelihood value found in all four Markov chains (the burn-in) were excluded, and the remaining trees were used to construct a majority rule consensus in PAUP (version 4.0b10; Swofford, 2002
). The percentages on this tree are the Bayesian posterior probabilities.
RESULTS
Structure, size, and composition of ITS
Length of the entire ITS region, including ITS1, 5.8S rDNA, and ITS2, varied among the Aglaia DNAs from 650 to 662 base pairs (bp; Table 1). For 5.8S rDNA, most Aglaia species exhibited a length of 164 bp (163 bp in A. silvestris (M. Roemer) Merrill; 168 bp in the clade formed by A. odorata Lour., A. pachyphylla Miq., and A. sapindina (F. von Muell.) Harms; 172 bp in A. coriacea). In Aglaieae (Aglaia excluded), the length of the entire ITS region varied from 649 to 660 bp (Table 2). In the three genera of Guareae, the length of the entire ITS region was slightly shorter and varied from 639 to 651 bp (Table 2). For the outgroup taxa (Melieae), the length of the entire ITS region was shorter than in the ingroup and varied from 639 to 645 bp (Table 1). Sequences of all ingroup and outgroup taxa contained the conserved angiosperm motif GGCRY—(4 to 7 n)— GYGYCAAGGAA at the ITS1 region (Liu and Schardl, 1994
), the conserved (C1–C6) and variable (V1–V6) domains determined for plant ITS2 sequences (Hershkovitz and Zimmer, 1996
), and the conserved angiosperm motif 5'-GAATTGCAGAATCC-3' within the 5.8S rDNA gene (Jobes and Thien, 1997
). Alignment of all ITS region sequence positions resulted in a matrix of 741 characters for the whole set of taxa. The length of the total matrix including 18S and 26S rDNA flanking regions was 947 characters.
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Analysis of ITS
Maximum parsimony analysis of the entire ITS region, comprising ITS1, 5.8S, and ITS2, produced 234 shortest trees of 1336 steps, CI of 0.44 and RI of 0.72 (Table 3). In the bootstrap consensus tree (tree not shown) tribe Aglaieae are monophyletic (BP 86), and Aglaia is paraphyletic as in the combined analysis. Representatives of the Amoora core group are strongly supported (BP 100) and sister (BP 56) to Lansium/Reinwardtiodendron (BP 100). Aglaia lawii and A. teysmanniana form a strongly supported clade (BP 100). As in the combined analysis, Aphanamixis borneensis and A. polystachya are related to Sphaerosacme decandra (BP 100); the relationship to A. sumatrana is unresolved.
Analysis of rps16 intron
Maximum parsimony analysis of partial rps16 intron sequences produced more than 15 000 shortest trees of 113 steps, CI of 0.85 and RI of 0.93 (Table 3). Due to the low level of variation and poor resolution in the strict consensus tree, we do not illustrate a tree here. Relative to just the ITS results, the addition of the rps16 intron data in the combined analyses did not alter topological patterns noticeably.
Combined Bayesian analysis
In the combined ITS/rps16 intron Bayesian tree (Fig. 2; burn in of 900 trees) Aglaieae are monophyletic (posterior probability, PP, 100), and Aglaia is not supported as monophyletic. Section Aglaia is monophyletic (PP 100), and section Amoora is unresolved with respect to section Aglaia and Lansium/Reinwardtiodendron. The clade formed by the intermediate species Aglaia lawii and A. teysmanniana is well supported (PP 100), as is that formed by representatives of the core group of section Amoora (PP 100) and Lansium/Reinwardtiodendron (PP 100). Again, Aphanamixis borneensis and A. polystachya are related to Sphaerosacme decandra (PP 100), and the relationship to A. sumatrana remains unclear.
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Relationships of Aglaia to other Aglaieae
Comparison of morphological and anatomical features to DNA data
Lansium as defined by Pennington and Styles (1975)
is distinguished from Aglaia by its simple indumentum, pentalocular ovary (rarely to be found in Aglaia), and the structure of its style and style head. However, the secondary xylem of Lansium was found to be indistinguishable from that of some species of Aglaia. Pennington and Styles (1975)
also found that Aglaia section Lansium of Kostermans (1966)
was heterogeneous and that those species for which floral characters were known could be confidently placed in either Lansium or Reinwardtiodendron. Aphanamixis is morphologically similar to Aglaia, Lansium, and Reinwardtiodendron (Pennington and Styles, 1975
). The embryo of Aphanamixis has cotyledons that are fused throughout their length, a condition unknown elsewhere in the subfamily except in Sphaerosacme. Except for the apparent unity of the cotyledons (a feature not investigated in all Aglaia species so far), there is no other macroscopic character that reliably separates Aphanamixis from Aglaia. Sphaerosacme has morphological similarities to both Reinwardtiodendron and Aphanamixis; it possesses the floral characters of the first and the fruit and seed characters of the latter (Pennington and Styles, 1975
). Sphaerosacme [former L. decandrum (Wall.) Harms], elevated to generic rank by Pennington and Styles (1975)
, is morphologically less closely related to Lansium and differs from it in floral, fruit, and seed characters. Sphaerosacme differs from Aglaia further in sepal, anther, style head, and radicle characters. The results of our study based on DNA data reflect the close relationships and reticulate morphological patterns observed among the genera of tribe Aglaieae. Aglaia is paraphyletic; Lansium and Reinwardtiodendron are most closely related to each other and to the core group of A. sect. Amoora (Figs. 1, 2). Aphanamixis, of which all three currently recognized species were included in our study, is potentially polyphyletic; its relations to the monotypic genus Sphaerosacme remain unclear (Figs. 1, 2). From a practical point of view, final decisions on broad-scale systematic reclassification necessary within Aglaieae should not be carried out until sequences of the remaining taxa of genera Lansium and Reinwardtiodendron (two and five species, respectively) are available. Unfortunately we were unable to amplify further species of these two genera up to now. Systematic reclassification of the whole tribe Aglaieae is beyond the scope of this paper, but it clearly will be a future aim of our continuing studies on Melioideae.
Concerning the relations of Aglaia to Lansium and Reinwardtiodendron, we propose to keep Lansium and Reinwardtiodendron as genera separate from Aglaia for the following reasons: (1) the unique occurrence of biologically highly active flavaglines in Aglaia in contrast to all other members of Meliaceae (Greger et al., 2001
), (2) differences in morphological and anatomical characteristics (no simple indumentum on vegetative parts of Aglaia plants; pentalocular ovary in Lansium and Reinwardtiodendron vs. usually uni- to trilocular ovary in Aglaia; Mabberley et al., 1995
; Pannell, 1992
; Pennington and Styles, 1975
), and (3) most importantly the low support for the clades uniting Lansium, Reinwardtiodendron, and the Amoora core group in the MP analyses (Fig. 1, BP 57).
Sectional classification in Aglaia
The type of the formerly recognized genus Amoora Roxb., Amoora cucullata Roxb., differs from typical Aglaia in several morphological characters (e.g., ovary and fruit condition and number of petals and anthers), but since this species was described, other, less extreme species have been discovered that provide morphological intermediates, so that recognition of Amoora appeared unacceptable to Pennington and Styles (1975)
. Finally, Pannell (1992)
recognized two sections within Aglaia, section Aglaia and section Amoora, and included species with intermediate characters in the latter. Our study shows that the sections as presently defined (on fruit dehiscence) may be untenable cladistically (Figs. 1, 2). Here we propose to recognize a third section in Aglaia: Neoaglaia (for a formal description, see end of discussion), comprising Aglaia teysmanniana, A. lawii, and A. beccarii C. de Candolle (a former synonym of A. lawii; Pannell, 2004
). This decision is supported by several lines of evidence:
; Fig. 2). Aglaia australiensis C. M. Pannell, A. meridionalis C. M. Pannell, and A. spectabilis (Miq.) Jain & Bennet belong to the flavagline chemotype, with A. spectabilis clearly differing due to the accumulation of less frequent benzofurans. Aglaia australiensis and A. meridionalis are the only accessions producing small amounts of unidentified flavonoids (Brem, 2002
). Aglaia lawii belongs to the bisamide chemotype, characterized by a tendency to accumulate bisamides. Individuals of Aglaia teysmanniana form another distinct group marked by exclusive accumulation of terpenoids (terpenoid chemotype; Brem, 2002
). Finally, the marker bisamide aglairubine was found in members of the core group of Amoora (Aglaia australiensis, A. meridionalis, and A. spectabilis) but not in former members of Amoora with intermediate flowers, Aglaia teysmanniana and A. lawii (Teichmann, 2002
; Fig. 2), here assigned to Neoaglaia.
Species types in Aglaia
Pannell (1992)
recognized "taxonomically isolated species," "variable," and "complex species." Of the seven complex species in the genus, one is recognized in section Amoora (Aglaia lawii) and six in section Aglaia (A. edulis (Roxb.) Wall., A. elaeagnoidea (A. Juss.) Benth., A. elliptica, A. korthalsii, A. leptantha, and A. tomentosa). We included all but one (Aglaia leptantha) currently recognized complex species of both sections in our analyses. For three (A. lawii, A. edulis, and A. tomentosa) of the seven complex species, we included between three and four individuals from different locations. This provides the possibility for a first assessment of the genetic circumscription of these species. For A. tomentosa (section Aglaia), individuals from different localities appear in different clades (Figs. 1, 2). For A. lawii and A. edulis (section Amoora), individuals from different localities exhibit genetic heterogeneity as well. Aglaia tomentosa is polyphyletic (Figs. 1, 2); A. lawii and A. edulis are either monophyletic (Fig. 1) or paraphyletic (Fig. 2). These findings based on macromolecular evidence of ITS and the rps16 intron are supported by chemotaxonomic characters. In Aglaia lawii, chemical heterogeneity and chemodiversity in individuals from different locations was observed by means of high performance liquid chromatography (HPLC) and gas chromatography coupled to mass spectrometry (GC-MS), displaying qualitative variation of major compounds (Brem, 2002
). Similar chemical heterogeneity was also found in A. tomentosa and A. edulis. According to the chemical profiles of A. tomentosa, different individuals from several locations can even be assigned to three different chemotypes (flavagline type, lignan type, terpenoid type; Brem, 2002
).
In addition to the complex species discussed, we included 16 variable species in addition to 13 taxonomically isolated species in our analyses. For eight of the 16 variable species, we included between two and five specimens from different locations. Within two species (A. sexipetala, A. teysmanniana), we found genetic heterogeneity similar to that in the complex species (Figs. 1, 2). Although variation among accessions of what are considered complex or variable species by Pannell (1992)
may mean that there are multiple biological species being lumped together, this is not the only conclusion that could be reached. If the accessions of a complex species do not form a clade (A. tomentosa), this could mean that further study may lead to the recognition of additional species, but it could also be a result of hybridization. The ITS is subject to "capture" through gene conversion, and it often is converted in the direction of the maternal parent (Chase et al., 2003
), so agreement of phylogenetic patterns obtained from analyses of ITS and the rps16 intron do not necessarily refute hybridization as the cause of such patterns (Chase et al., 2003
). Another possible explanation for the patterns observed could be the independent evolution of morphologically convergent characters on the two sides of Wallace's line. This could be the reason for morphological similarity, but genetic non-relatedness in A. tomentosa, in which the accessions from Thailand (west of Wallace's line) and the accession from Australia (east of Wallace's line) appear in different parts of the trees (Figs. 1, 2). We favor the explanation of morphological convergence over hybridization because (1) intermediates are not usually found in the same locality as the two taxa between which they are intermediate (C. M. Pannell, personal observation), and (2) all accessions of section Aglaia (except A. elaeagnoidea) collected on the eastern side of Wallace line form a moderately to strongly supported clade (compare top clades in Figs. 1, 2; BP 83, PP 100, BP 70), including the Australian accession of A. tomentosa. The fact that Aglaia tomentosa is one of the most widespread and morphologically diverse complex species in Aglaia, covering most of the total distribution range of the genus, will make this taxon an appropriate model for evaluating the usefulness of currently used diagnostic characters like the indumentum (structure, size, distribution of trichomes on different parts of the plant) for delimitation of species in Aglaia and other Meliaceae in our future studies.
Concluding remarks
Investigations of DNA data and secondary metabolites of Aglaia and related genera contribute significantly to a better understanding of the intricate systematic relationships of this group of trees that constitute an important component of the moist tropical forest in the Indomalesian region. This study is the first to assess the current circumscription of Aglaieae, Aglaia and its sections, and species concepts with data independent of morphology. Maximum parsimony and Bayesian analyses of nuclear ITS and plastid rps16 intron, as well as comparison of chemical profiles observed by means of HPLC and GC-MS, confirm close relationships among the genera of tribe Aglaieae and provide helpful tools for infrageneric delimitation of sections in Aglaia. Concerning the relations of Aglaia to Lansium and Reinwardtiodendron, we propose to keep Lansium and Reinwardtiodendron as genera separate from Aglaia, at least for the time being. Within Aglaia itself, we recognize three different taxonomic units: section Aglaia, comprising members of the present section Aglaia, a new section Neoaglaia, comprising morphological intermediates between Aglaia and Amoora, and section Amoora, comprising the core group of Amoora, now excluding morphological intermediates between the former two sections. These taxonomic decisions are based on DNA data, secondary metabolites, as well as morphological variation. Furthermore, macro- and micromolecular data indicate that variable and complex species are more heterogeneous, i.e., probably containing more than one taxon each, than taxonomically isolated species. Aglaia tomentosa, one of the most widespread and morphologically diverse complex species in Aglaia, will serve as model taxon for evaluating the usefulness of currently used diagnostic characters in Meliaceae in our future studies.
A third section in Aglaia
Section Neoaglaia Harms in A. Engler and K. Prantl, Die Natuerlichen Pflanzenfamilien, III 4: 300 (1896); Harms in H. Harms and J. Mattfeld, Die Natuerlichen Pflanzenfamilien, ed. 2, 19 bI: 146 (1940). Lectotype species (designated here): Aglaia teysmanniana (Miq.) Miq., Annales Musei Botanici Lugduno-Batavi 4: 48–49 (1868).
Petals 3–5 (–6), anthers (5 or) 6–10; fruit small 1–2.8 (–6) x 1.2–2.3 (–3.5) cm, dehiscent; pericarp pink (sometimes carmine red or yellow in Aglaia lawii).
Additional species
Aglaia lawii (Wight) Saldanha ex Ramamoorthy and A. beccarii C. de Candolle. Aglaia beccarii was recently removed from synonymy with A. lawii (Pannell, 2004
). Aglaia beccarii is almost confined to Borneo, there being one record from the Philippines.
Notes
Section Neoaglaia differs from section Amoora in the variable number of petals and the color of the pericarp. In Amoora there are three petals, or rarely two in Aglaia meridionalis, and the pericarp is yellow, red, orange, or reddish-brown. The mature fruit of most species in section Amoora is large, 6 cm or more long and 5 cm or more wide (2.5–5.5 cm wide in the long, narrow fruits of Aglaia flavida Merrill & Perry). The fruits of some Australasian species are smaller. In Aglaia australiensis (endemic to Australia), A. lepidopetala Harms (endemic to New Guinea) and A. meridionalis (endemic to Australia), they are 2.5–4 x 2.5–3 cm.
FOOTNOTES
1 The authors thank Tod F. Stuessy, Head of the Dept. of Higher Plant Systematics and Evolution, University of Vienna, for the facilities provided; Annette W. Coleman, Brown University, USA, for her advice on alignment of ITS sequences based on secondary structure; and Brigitte Brem, University of Vienna, Austria, for allowing us to use data on secondary metabolites in Aglaia from her Ph.D. thesis. Financial support for this study was provided by the FWF to Rosabelle Samuel (grant no. P14150-BOT). 
6 Present address: Jodrell Laboratory, Section of Molecular Systematics, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3DS, UK (alexandra.muellner{at}univie.ac.at
, a.muellner{at}kew.org
.)
LITERATURE CITED
Agostinho S. M. M M. F. das G. F da Silva J. B Fernandes P. C Vieira A. L Pinheiro E. F Vilela 1994 Limonoids from Toona ciliata and speculations on their chemosystematic and ecological significance. Biochemical Systematics and Ecology 22: 323-328[CrossRef]
Bacher M O Hofer G Brader S Vajrodaya H Greger 1999 Thapsakins: possible biogenetic intermediates towards insecticidal cyclopenta[b]benzofurans from Aglaia edulis. Phytochemistry 52: 253-263[CrossRef][ISI]
Baldwin B. G S Markos 1998 Phylogenetic utility of the external transcribed spacer (ETS) of 18S–26S rDNA: congruence of ETS and ITS trees of Calycadenia (Compositae). Molecular Phylogenetics and Evolution 10: 449-463[CrossRef][ISI][Medline]
Baldwin B. G M. J Sanderson J. M Porter M. F Wojciechowski C. S Campbell M. J Donoghue 1995 The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247-277[CrossRef][ISI]
Blume C. L 1825 Bijdragen tot de flora van Nederlandsch Indië, 4, 169– 172. Batavia
Brader G S Vajrodaya H Greger M Bacher H Kalchhauser O Hofer 1998 Bisamides, lignans, triterpenes, and insecticidal cyclopenta[b]benzofurans from Aglaia species. Journal of Natural Products 61: 1482-1490[CrossRef][Medline]
Brem B 2002 Distribution and insecticidal properties of characteristic plant constituents from tropical Aglaia and Stemona species. Ph.D. thesis, University of Vienna, Austria
Chase M. W H. G Hills 1991 Silica gel: an ideal desiccant for preserving field-collected leaves for use in molecular studies. Taxon 40: 215-220[CrossRef][ISI]
Chase M. W S Knapp J Clarkson A. V Cox J Joseph I. Y Butsko J. A Marshall V Savolainen A. S Parokonny 2003 Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae). Annals of Botany 92: 107-127
Clarkson J. J M. W Chase M. M Harley 2002 Phylogenetic relationships in Burseraceae based on plastid rps16 intron sequences. Kew Bulletin 57: 183-193[CrossRef]
Cui B H Chai T Santisuk V Reutrakul N. R Farnsworth G. A Cordell J. M Pezzuto A. D Kinghorn 1997 Novel cytotoxic 1H-cyclopenta[b]benzofuran lignans from Aglaia elliptica. Tetrahedron 53: 17625-17632[CrossRef][ISI]
Da Silva M. F. das G. F O. R Gottlieb D. L Dreyer 1984 Evolution of limonoids in the Meliaceae. Biochemical Systematics and Ecology 12: 299-310
Da Silva M. F. das G. F S. M. M Agostinho J. R de Paula J. O Neto I Castro-Gamboa E. R Filho J. B Fernandes P. V Vieira 1999 Chemistry of Toona ciliata and Cedrela odorata graft (Meliaceae): chemosystematic and ecological significance. Pure and Applied Chemistry 71: 1083-1087[CrossRef][ISI]
De Candolle C 1878 Amoora & Aglaia. In A. and C. de Candolle [eds.], Monographiae Phanerogamarum I, 579–592, 601–628. Paris, France
Doyle J. J J. L Doyle 1987 A rapid DNA isolation procedure from small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15
Dreyer M B. W Nugroho F. I Bohnenstengel R Ebel V Wray L Witte G Bringmann J Mühlbacher M Herold P. D Hung L. C Kiet P Proksch 2001 New insecticidal rocaglamide derivatives and related compounds from Aglaia oligophylla. Journal of Natural Products 64: 415-420[CrossRef][Medline]
Edwards K. J P. A Gadek 2001 Evolution and biogeography of Alectryon (Sapindaceae). Molecular Phylogenetics and Evolution 20: 14-26[CrossRef][ISI][Medline]
Engelmeier D F Hadacek T Pacher S Vajrodaya H Greger 2000 Cyclopenta[b]benzofurans from Aglaia species with pronounced antifungal activity against rice blast fungus (Pyricularia grisea). Journal of Agricultural and Food Chemistry 48: 1400-1404[CrossRef][ISI][Medline]
Felsenstein J 1985 Confidence limits on phylogenetics: an approach using the bootstrap. Evolution 39: 783-791[CrossRef][ISI]
Fitch W. M 1971 Toward defining the course of evolution: minimal change for a specific tree topology. Systematic Zoology 20: 406-416[CrossRef][ISI]
Fuzzati N W Dyatmiko A Rahman F Achmad K Hostettmann 1996 Triterpenoids, lignans and a benzofuran derivative from the bark of Aglaia elaeagnoidea. Phytochemistry 42: 1395-1398[CrossRef][ISI]
Greger H T Pacher B Brem M Bacher O Hofer 2001 Insecticidal flavaglines and other compounds from Fijian Aglaia species. Phytochemistry 57: 57-64[CrossRef][ISI][Medline]
Greger H T Pacher S Vajrodaya M Bacher O Hofer 2000 Infraspecific variation of sulfur-containing bisamides from Aglaia leptantha. Journal of Natural Products 63: 616-620[CrossRef][Medline]
Harms H 1896 Meliaceae. In A. Engler and K. Prantl [eds.], Die natuerlichen Pflanzenfamilien III, vol. 4, 300, 258–308. W. Engelmann, Leipzig, Germany
Harms H 1940 Meliaceae. In H. Harms and J. Mattfeld [eds.], Die natuerlichen Pflanzenfamilien, 2nd ed., 19 bI., 1–172. W. Engelmann, Leipzig, Germany
Hershkovitz M. A E. A Zimmer 1996 Conservation patterns in angiosperm rDNA ITS2 sequences. Nucleic Acids Research 24: 2857-2867
Huelsenbeck J. P F. R Ronquist 2001 MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755
IUCN [World Conservation Union]. 2003 2003 IUCN Red List. Available at website, http://www.redlist.org/
Jobes D. V L. B Thien 1997 A conserved motif in the 5.8S ribosomal RNA (rRNA) gene is a useful diagnostic marker for plant internal transcribed spacer (ITS) sequences. Plant Molecular Biology Reporter 15: 326-334[CrossRef]
King G 1895 Materials for a flora of the Malayan Peninsula. Journal of the Asiatic Society of Bengal 64: 51-80
Kostermans A. J. G. H 1966 A monograph of Aglaia sect. Lansium Kosterm. (Meliaceae). Reinwardtia 7: 221-282
Liu J.-S C. L Schardl 1994 A conserved sequence in internal transcribed spacer 1 of plant nuclear rRNA genes. Plant Molecular Biology 26: 775-778[CrossRef][ISI][Medline]
Mabberley D. J 1985 Florae Malesianae Praecursores LXVII. Meliaceae (divers genera). Blumea 31: 129-152
Mabberley D. J C. M Pannell A. M Sing 1995 Meliaceae. Flora Malesiana series I, vol. 12, part 1. Rijksherbarium/Hortus Botanicus, Leiden University, Leiden, Netherlands
Mathews D. H J Sabina M Zuker D. H Turner 1999 Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology 288: 911-940[CrossRef][ISI][Medline]
Miquel F. A. G 1868 Aglaia. In F. A. G. Miquel [ed.], Monographia Meliacearum Archipelagi indici, Annales Musei Botanici Lugduno-Batavi 4: 38-58 C. G. Van der Post, Amsterdam, Netherlands
Mohamad K M.-T Martin H Najdar C Gaspard T Sévenet K Awang H Hadi M Païs 1999 Cytotoxic 3,4-secoapotirucallanes from Aglaia argentea bark. Journal of Natural Products 62: 868-872[CrossRef][Medline]
Muellner A. N R Samuel S. A Johnson M Cheek T. D Pennington M. W Chase 2003 Molecular phylogenetics of Meliaceae based on nuclear and plastid DNA sequences. American Journal of Botany 90: 471-480
Mulholland D. A M Kotsos H. A Mahomed D. A. H Taylor 1998 Triterpenoids from Owenia cepiodora. Phytochemistry 49: 2457-2460[CrossRef][ISI]
Neto J. O M. F. das G. F da Silva E. R Fo J. B Fernandes P. C Vieira A. L Pinheiro 1998 Norlimonoids from seeds of Toona ciliata. Phytochemistry 49: 1369-1373[CrossRef][ISI]
Nugroho B. W R. A Edrada V Wray G Bringmann M Gehling P Proksch 1999 An insecticidal rocaglamide derivatives and related compounds from Aglaia odorata (Meliaceae). Phytochemistry 51: 367-376[CrossRef][ISI]
Oxelman B M Lidén D Berglund 1997 Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Systematics and Evolution 206: 393-410[CrossRef][ISI]
Palumbi S. R 1996 Nucleic acids II: the polymerase chain reaction. In D. M. Hillis, C. Moritz, and B. K. Mable [eds.], Molecular systematics, 205–247. Sinauer Associates, Sunderland, Massachusetts, USA
Pannell C. M 1992 A taxonomic monograph of the genus Aglaia Lour. (Meliaceae). Kew Bulletin Additional Series XVI. HMSO, London, UK
Pannell C. M 1993 A monograph of Aglaia (Meliaceae)—a correction. Kew Bulletin 48: 244[CrossRef]
Pannell C. M 1998a Taxonomy, ecology and reproductive biology of Aglaia (Meliaceae). In H. C. F. Hopkins, C. R. Huxley, C. M. Pannell, G. T. Prance, and F. White [eds.], The biological monograph: the importance of field studies and functional syndromes for taxonomy and evolution of tropical plants, a Festschrift for Frank White, 59–77. Royal Botanic Gardens, Kew, London, UK
Pannell C. M 1998b Species delimitation in Aglaia. In H. C. F. Hopkins, C. R. Huxley, C. M. Pannell, G. T. Prance, and F. White [eds.], The biological monograph, the importance of field studies and functional syndromes for taxonomy and evolution of tropical plants, a Festschrift for Frank White, 124–127. Royal Botanic Gardens, Kew, London, UK
Pannell C. M 2004 Three new species, two new subspecies and five new combinations at the subspecific level in Aglaia Lour. (Meliaceae). Kew Bulletin 59: 87-94[CrossRef]
Pellegrin F 1911 Sur les genres Aglaia, Amoora et Lansium. Notulae Systematicae (Phanerogamie) 1: 284-290
Pennington T. D B. T Styles 1975 A generic monograph of the Meliaceae. Blumea 22: 419-540
Popp M B Oxelman 2001 Inferring the history of the polyploid Silene aegea (Caryophyllaceae) using plastid and homoeologous nuclear DNA sequences. Molecular Phylogenetics and Evolution 20: 474-481[CrossRef][ISI][Medline]
Posada D K. A Crandall 1998 Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817-818
Posada D K. A Crandall 2001 Selecting the best-fit model of nucleotide subsitution. Systematic Biology 50: 580-601[ISI][Medline]
Puripattanavong J S Weber V Brecht A. W Frahm 2000 Phytochemical investigation of Aglaia andamanica. Planta Medica 66: 740-745[CrossRef][ISI][Medline]
Saifah E J Puripattanavong K Likhitwitayawuid G. A Cordell H Chai J. M Pezzuto 1993 Bisamides from Aglaia species: structure analysis and potential to reverse drug resistance with cultured cells. Journal of Natural Products 56: 473-477[CrossRef][Medline]
Saifah E R Suttisri S Shamsub T Pengsuparp V Lipipun 1999 Bisamides from Aglaia edulis. Phytochemistry 52: 1085-1088[CrossRef][ISI][Medline]
Salamin N M. W Chase T. R Hodkinson V Savolainen 2003 Assessing internal support with large phylogenetic DNA matrices. Molecular Phylogenetics and Evolution 27: 528-539[CrossRef][ISI][Medline]
Sun Y D. Z Skinner G. H Liang S. H Hulbert 1994 Phylogenetic analysis of Sorghum and related taxa using transcribed spacers of nuclear ribosomal DNA. Theoretical and Applied Genetics 89: 26-32[ISI]
Swofford D. L 2002 PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4. Sinauer Associates, Sunderland, Massachusetts, USA
Taylor D. A. H 1981 Chemotaxonomy. The occurrence of limonoids in the Meliaceae. In T. D. Pennington, B. T. Styles, and D. A. H. Taylor [eds.], Meliaceae, Flora Neotropica, Monograph 28, 450–459. The New York Botanical Garden, Bronx, New York, USA
Teichmann K 2002 Comparative phytochemical analyses of Aglaia species from section Amoora (Meliaceae). M.Sc. thesis, University of Vienna, Vienna, Austria
Thompson J. D T. J Gibson F Plewniak F Jeanmougin D. G Higgins 1997 The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876-4882
Vaasen A D Begerow U Lüttge R Hampp 2002 The genus Clusia L.: molecular evidence for independent evolution of photosynthetic flexibility. Plant Biology 4: 86-93[CrossRef][ISI]
Wang B.-G R Ebel B. W Nugroho D Prijono W Frank K. G Steube X.-J Hao P Proksch 2001 Aglacins A-D, first representatives of a new class of aryltetralin cyclic ether lignans from Aglaia cordata. Journal of Natural Products 64: 1521-1526[CrossRef][Medline]
Wanntorp L H. E Wanntorp B Oxelman M Källersjö 2001 Phylogeny of Gunnera. Plant Systematics and Evolution 226: 85-107[CrossRef][ISI]
Weber S J Puripattanavong V Brecht A. W Frahm 2000 Phytochemical investigation of Aglaia rubiginosa. Journal of Natural Products 63: 636-642[CrossRef][Medline]
White T. J T Bruns S Lee J Taylor 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White [eds.], PCR protocols: a guide to methods and applications, 315–322. Academic Press, San Diego, California, USA
Whitten W. M N. H Williams M. W Chase 2000 Subtribal and generic relationships of Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: combined molecular evidence. American Journal of Botany 87: 1842-1856
Zuker M D. H Mathews D. H Turner 1999 Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In J. Barciszewski and B. F. C. Clark [eds.], RNA biochemistry and biotechnology, 11–43. NATO ASI Series. Kluwer Academic Publishers, Dordrecht, Netherlands
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