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471
American Journal of Botany 90(3): 471–480. 2003.
M
OLECULAR PHYLOGENETICS OF
M
ELIACEAE
(S
APINDALES
)
BASED ON NUCLEAR AND PLASTID
DNA
SEQUENCES
1
A
LEXANDRA
N. M
UELLNER
,
2,5
R
OSABELLE
S
AMUEL
,
2
S
HEILA
A. J
OHNSON
,
3
M
ARTIN
C
HEEK
,
4
T
ERENCE
D. P
ENNINGTON
,
4
AND
M
ARK
W. C
HASE
3
2
Botanisches Institut und Botanischer Garten, Universitaet Wien, Rennweg 14, A-1030 Vienna, Austria;
3
Jodrell Laboratory,
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK; and
4
The Herbarium, Royal Botanic Gardens, Kew,
Richmond, Surrey TW9 3AB, UK
Phylogenetic analyses of Meliaceae, including representatives of all four currently recognized subfamilies and all but two tribes (32
genera and 35 species, respectively), were carried out using DNA sequence data from three regions: plastid genes rbcL, matK (partial),
and nuclear 26S rDNA (partial). Individual and combined phylogenetic analyses were performed for the rbcL, matK, and 26S rDNA
data sets. Although the percentage of informative characters is highest in the segment of matK sequenced, rbcL provides the greatest
number of informative characters of the three regions, resulting in the best resolved trees. Results of parsimony analyses support the
recognition of only two subfamilies (Melioideae and Swietenioideae), which are sister groups. Melieae are the only tribe recognized
previously that are strongly supported as monophyletic. The members of the two small monogeneric subfamilies, Quivisianthe and
Capuronianthus, fall within Melioideae and Swietenioideae, respectively, supporting their taxonomic inclusion in these groups. Fur-
thermore, the data indicate a close relationship between Aglaieae and Guareeae and a possible monophyletic origin of Cedreleae of
Swietenioideae. For Trichilieae (Melioideae) and Swietenieae (Swietenioideae) lack of monophyly is indicated.
Key words: matK; Meliaceae; nuclear and plastid DNA; phylogeny; rbcL; 26S rDNA; Sapindales.
Meliaceae are a woody family widely distributed throughout
the tropics and subtropics, with only slight penetration into
temperate zones; they occur in a variety of habitats from rain
forests and mangrove swamps to semi-deserts. The timbers of
certain Meliaceae are some of the most sought after in the
world, such that natural stands have been much depleted
(Keay, 1996; Plumptree, 1996; Rodan and Campbell, 1996;
Snook 1996; Verissimo et al., 1998; Laurance, 1999; O’Neill
et al., 2001). Other uses of Meliaceae comprise shade and
street trees, fruit trees, and, last but not least, sources of bio-
logically active compounds (reviewed in Mabberley et al.,
1995). Among other secondary metabolites, Meliaceae synthe-
size and accumulate bitter and biologically active nortriterpen-
oids, which are also known as limonoids and meliacins. These
and other compounds have aroused considerable commercial
interest due to their insect-antifeedant (e.g., Abdelgaleil et al.,
2001; Simmonds et al., 2001), insect-repellent (e.g., Shukla,
et al., 1997; Valladares et al., 1999) or/and insecticidal (e.g.,
Schneider et al., 2000; Greger et al., 2001), molluscicidal (e.g.,
Pina et al., 1998; Singh et al., 1998), antifungal (e.g., Govin-
dachari et al., 1999; Engelmeier et al., 2000), bactericidal (e.g.,
Kumar and Gopal, 1999; Aboutabl et al., 2000), and plant-
antiviral (e.g., Singh et al., 1988) activities as well as their
numerous medicinal effects in humans and animals (e.g., Bam-
1
Manuscript received 12 March 2002; revision accepted 15 October 2002.
The authors thank T. F. Stuessy, Head of the Dept. of Higher Plant System-
atics and Evolution, University of Vienna, for the facilities provided, N. Gun-
atilleke and R. Ratnayake, Dept. of Botany, University of Peradeniya, for their
support to collect material in Sri Lanka, and D. J. Mabberley, Royal Botanic
Gardens Sydney, for his valuable comments on the manuscript. Financial sup-
port for this study was provided by the FWF to Rosabelle Samuel (grant no.
P14150-BOT) and the Royal Botanic Gardens, Kew, for providing an intern-
ship to SAJ.
5
Author for reprint requests (e-mail: alexandra.muellner@univie.ac.at).
ba et al., 1999; Benencia et al., 2000; Benosman et al., 2000).
The limonoids (or meliacins) might offer possibilities for in-
frafamiliar taxonomy (e.g., Taylor in Pennington et al., 1981;
Mulholland et al., 1998), but our knowledge is still extremely
fragmentary (Mabberley et al., 1995) so that comprehensive
phylogenetic conlusions (Da Silva et al., 1984, 1999; Agostin-
ho et al., 1994; Neto et al., 1998) have been premature and
often conflicting.
Compared to other groups of similar size, Meliaceaecontain
a relatively wide range of floral, fruit, and seed morphologies.
For example, within Aglaia alone inflorescences can vary from
one-third to two-thirds of a meter long with profuse branching
and abundant flowers to a much reduced few-flowered inflo-
rescence 1–2 cm long. Seeds of Meliaceae are some of the
most diverse and intricate in structure so far investigated
(Mabberley et al., 1995). They are usually pendulous and ep-
itropous (but apotropous in the Australian Synoum, Guareae)
in relation to the placenta. They are usually anatropous (but
hemi-anatropous in most Turraeeae and Cipadessa), occasion-
ally orthotropous as in all Chisocheton and some Guarea spe-
cies, and campylotropous in Nymania (Turraeeae), for exam-
ple. In general, a diversity of primitive morphological char-
acters can be observed side-by-side with an array of derived
ones, but these are typically connected by intermediates. For
this reason, the family has been a source of systematic diffi-
culty, as the taxonomic history of the group clearly shows
(summary in Pennington and Styles, 1975). Until the generic
monograph of the family by Pennington and Styles (1975),
there was persistent disagreement as to the number of genera
and their circumscriptions and the best way to accommodate
them in tribes and subfamilies. This uncertainty may have
been due to the diffuse and often reticulate nature of variation
that seems to have been based on several parallel evolutionary
trends occurring in flowers and fruits independently (Penning-
472 [Vol. 90A
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J
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T
ABLE
1. Characters used to distinguish the subfamilies of Meliaceae sensu Pennington and Styles (1975).
Character Melioideae Quivisianthoideae Swietenioideae Capuronianthoideae
Buds
Fruits
Seeds
naked (not all)
loculicidal capsule, berry, drupe, nut
not corky, not winged
naked
loculicidal capsule
winged
with scale leaves
septifragal capsule
winged or corky
naked
septifragal capsule
corky
T
ABLE
2. Statistics for each of the maximum parsimony analyses (CI
5
consistency index; RI
5
retention index; GC
5
guanine and cytosine).
Data set matK rbcL 26S rDNA matK/rbcL matK/rbcL/26S rDNA
No. of taxa
No. of Meliaceae
No. of characters
No. of variable sites
No. of informative characters
Length of shortest tree (no. of steps)
No. of shortest trees
CI
RI
GC content
32
28
893
297
148
460
9700
0.69
0.84
36.20
44
38
1387
247
170
474
612
0.58
0.84
43.86
33
30
880
130
71
289
46
0.48
0.64
60.52
47
41
2280
544
318
934
9840
0.63
0.84
41.48
47
41
3160
674
389
1251
3559
0.57
0.79
46.15
ton and Styles, 1975). Thus, some genera and most tribes can
only be diagnosed by using a combination of several charac-
ters. Each character taken separately may occasionally be un-
reliable as a diagnostic tool.
Unlike some other tropical woody groups, Meliaceae also
exhibit a considerable range of chromosome numbers (Styles
and Vosa, 1971; review in Styles and Bennett, 1992). A base
number of n514 or multiples such as 28, 42, or even higher
ploidy represent, according to Khosla and Styles (1975), the
vestiges of an archaic stock of n57, which has been consid-
ered by many workers in the field as one of the original base
numbers of angiosperms (Stebbins, 1971). Differences in num-
ber have been observed between even putatively closely re-
lated species, e.g., within Swietenia,Cedrela, and Toona.At
the subfamilial and tribal levels, chromosome numbers would
appear to have no obvious pattern (Styles and Vosa, 1971).
Polyploid series are by no means a rare phenomenon in the
group, being apparent in Aphanamixis,Aglaia,Chisocheton,
Dysoxylum, and Swietenia, for example, including the occur-
rence of intraspecific chromosome races in some genera
(Khosla and Styles, 1975; Mabberley, et al., 1995). Extensive
hybridization accompanied by introgression may have oc-
curred in these groups, perhaps leading to the reticulate pat-
terns of morphological traits and the complex interrelation-
ships of the various taxa that are now encountered (Khosla
and Styles, 1975). Karyological data give a rather confusing
picture of the relationships between taxa, and although some
conclusions have been drawn, they have remained mostly
speculative (e.g., Mehra et al., 1972; Datta and Samanta,
1977).
The most authoritative work on Meliaceae is at present the
generic monograph by Pennington and Styles (1975); currently
recognized are 50 genera with about 575 species (Pennington
and Styles, 1975; Mabberley et al., 1995; Chase et al., 1999).
Pennington and Styles (1975) recognized four subfamilies;
Melioideae and Swietenioideae
6
consist of seven tribes with
35 genera and of three tribes with 13 genera, respectively.
6
Antedated by the obscure name Cedreloideae (D. J. Mabberley, personal
communication, Royal Botanic Gardens Sydney) which is in the course of
being proposed for formal rejection to the committee for spermatophytes of
IAPT.
Quivisianthoideae and Capuronianthoideae contain a single
monotypic genus each (Quivisianthe and Capuronianthus, re-
spectively). The last two subfamilies were newly recognized
by Pennington and Styles (1975). The following characters,
among others, were particularly important for the delimitation
of subfamilies (Table 1): bud scales (presence/absence), fruits
(fleshy/leathery or dry, loculicidal or septifragal dehiscence,
capsules, berries, drupes or nuts), and seeds (winged/not
winged, corky/not corky). Pennington and Styles (1975) stated
that only Swietenioideae have bud scales. Later studies have
shown that the presence of bud scales is not restricted to Swie-
tenioideae but can be also found in several representatives of
Melioideae (e.g., in some species of Turraea,Trichilia, and
Ruagea; Pennington et al., 1981; Cheek, 1990b). Melioideae
and Quivisianthe share loculicidal dehiscence of their capsules,
although in Melioideae capsules are only one out of four fruit
types and include also berries, drupes, and nuts. In Melioideae,
fruits are fleshy and seeds are never winged with a woody or
corky testa, but they usually have a fleshy arillode or sarco-
testa. In Quivisianthoideae, fruits are dry, and seeds are
winged with a dry, non-fleshy testa. Swietenioideae and Ca-
puronianthus share capsules with septifragal dehiscence. In
Capuronianthus, seeds are not fleshy or winged but have a
corky sarcotesta. In Swietenioideae, seeds are variously
winged but are not fleshy, and they have a woody or corky
sarcotesta.
To assess circumscription of the subfamilies and tribes pro-
posed by Pennington and Styles (1975) and infer phylogenetic
relationships among them, we performed parsimony analyses
of sequence data from three regions: plastid rbcL, matK, and
nuclear 26S rDNA. We included representatives of all four
currently recognized subfamilies and all but two tribes (32
genera and 35 species altogether). The phylogenetic utility of
26S rDNA sequence data in the angiosperms has recently been
demonstrated (e.g., Kuzoff et al., 1998; Asmussen et al.,
2000). Larson (1991) suggested that the difference in rates of
base substitution between conserved core regions and expan-
sion segments of 26S rDNA could be exploited for phyloge-
netic inference at different taxonomic levels; conserved core
regions could be used at higher taxonomic levels, and expan-
sion segments could be employed among more closely related
March 2003] 473M
UELLNER ET AL
.—M
OLECULAR PHYLOGENETICS OF
M
ELIACEAE
(S
APINDALES
)
T
ABLE
3. Statistics for transitions (ts) and transversions (tv) based on
optimizations on one of the trees from the combined maximum
parsimony analysis of all three genes (CI
5
consistency index, RI
5
retention index).
Data set matK rbcL 26S rDNA
No. of steps (ts/tv)
No. of ts
No. of tv
Ts/tv ratio
CI
RI
CI of ts
RI of ts
CI of tv
RI of tv
461
255
206
1.24
0.69
0.84
0.77
0.85
0.62
0.83
480
277
203
1.36
0.57
0.84
0.68
0.84
0.45
0.83
310
230
80
2.88
0.44
0.57
0.45
0.53
0.43
0.61
taxa. We sequenced in this study a roughly 1200 base-pair
fragment from the 59end of the gene, which includes four of
the expansion segments. The matK gene has been shown to
have one of the highest nucleotide substitution rates among 20
plastid genes greater than 1 kilobase (kb) in length, and it is
currently being used for inferring phylogenetic relationships at
the intrafamiliar level (e.g., Koch et al., 2001; Mort et al.,
2001). Because of the large data base of rbcL sequences avail-
able (nearly every family and nearly complete sets of genera
for some families) and because patterns of relationships and
support derived from rbcL data sets are not significantly worse
than commonly sequenced noncoding regions (Chase et al.,
2000), it makes sense to include rbcL as one of the regions
when sequencing more than one region (Chase and Albert,
1998).
Many recent studies have indicated that combined molecular
data using regions with different numbers of variable sites pro-
vide resolution at different taxonomic levels, and phylogenetic
resolution and levels of support are improved by directly com-
bining independent molecular data sets (e.g., Chase and Cox,
1998; Soltis et al., 1998, 1999, 2000; Qiu et al., 1999; Savo-
lainen et al., 2000a). Our approach here is to assess patterns
of support for each gene separately and, if no evidence of
incongruence is present, to combine all three in a single anal-
ysis as the basis for further discussion.
MATERIALS AND METHODS
Plant material—The taxa used in this study, their sample designation, and
GenBank accession numbers have been archived at the Botanical Society of
America website (http://ajbsupp.botany.org/v90/). We included 32 (out of the
50) genera and 35 species, comprising representatives of all four subfamilies
and all but two tribes.
Isolation of DNA and amplification—Total genomic DNA was isolated
using the procedure described by Doyle and Doyle (1987) except that most
DNA samples were purified by cesium chloride/ethidium bromide gradients
(1.55 g/mL). 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: 26S1., 26S2., and
1229REV,for 26S rDNA (Kuzoff et al., 1998), 390F.and 1326R,for
matK (Cue´noud et al., 2002) and 1F.and 1460R,for rbcL (Fay et al.,
1998). The fragment size amplified was between 870 and 1210 bp for 26S,
870 and 910 bp for matK, and 1436 and 1460 bp for rbcL (this variation in
length is due to the variable downstream position of the ribosomal control
site for which 1460R,was designed). A 100-mL reaction mix contained 62
mL ddH
2
O, 10 mL103reaction buffer, 8 mL 15 mmol MgCl
2
,8mL 10 mmol
dNTPs, 2 mL of the primers each (20 pmol), 2 mL template DNA (100–2000
ng/mL), 1 mL 1 unit/mL DNA polymerase (Promega, Mannheim, Germany),
as well as 5 mL dimethyl sulfoxide (DMSO) for nuclear 26S rDNA and 5 mL
bovine serum albumin (BSA; 0.4%) for plastid genes. These additives are
thought to stabilize the enzyme, reduce secondary structure problems, or favor
precise annealing (Palumbi, 1996). Amplifications were carried out using the
following program (with slight modifications for some accessions): initial de-
naturation for 3 min at 958C, followed by one cycle of denaturation for 1.5
min at 958C, annealing for 1 min at 458C and extension for 1 min at 728C,
followed by 36 cycles of denaturation for 1 min at 958C, annealing for 1 min
at 488C, and extension for 1 min at 728C. The amplification was completed
by holding the reaction mixture for 10 min at 728C to allow complete exten-
sion of the PCR products. After amplification, samples were gel purified using
a QIAquick gel extraction kit (Qiagen, Margaritella,Vienna, Austria).
Sequencing—The same primers as cited above were also used for sequencing.
For rbcL, four additional internal sequencing primers were used: 636F., 724R,
(Fay et al., 1998), F2N.(59-CCAAGTTGAGAGAGATAAATTGAACAAG-39)
and F2NN.(59-GCAAATACTAGCTTGGCTCATTATTGCCG-39), the last two
designed for this study. A 10-mL cycle sequencing reaction mix contained 7 mL
purified template DNA (100–400 ng/mL), 2 mL BigDye Terminator RR Mix
(Applied Biosystems, Vienna, Austria) and 1 mL 10-pmol primer. The follow-
ing program was used (GeneAmp PCR System 9700, Applied Biosystems):
denaturation for 10 s at 968C, annealing for 5 s at 508C, extension for 4 min
at 608C (25 cycles). To produce betterquality 26S rDNA sequences, we added
2% DMSO to the cycle sequencing reactions; DMSO breaks down secondary
structure formed by guanine- and cytosine-rich regions and thereby produces
longer lengths of readable sequence. The templates were sequenced on an
ABI PRISM 377 DNA Sequencer (ABI) using dye terminators following pro-
tocols provided by the manufacturer (ABI).
Sequence editing and alignment—For editing, the software programs Au-
toassembler version 1.4.0 (Applied Biosystems) and DNA STRIDER version
1.2 (Christian Marck, CEA—Commissariat a`L’e`nergie Atomique/Saclay,
France) were used. Alignment of sequences was done by eye following the
recommendation of Kelchner (2000). A total of 893, 1387, and 880 nucleo-
tides were included in the matrices for phylogenetic analyses for matK, rbcL,
and 26S rDNA, respectively. Gaps were coded as missing data (there were
not many gaps within these taxa, so alignment was a simple matter). These
sequences have been deposited in GenBank under the accession numbers
AY128144-AY128252 (http://www.ncbi.nlm.nih.gov/); the combined matrix is
available from A. N. Muellner (alexandra.muellner@univie.ac.at) and M. W.
Chase (m.chase@rbgkew.org.uk).
Phylogenetic analysis—Individual and combined parsimony analyses of the
26S, matK, and rbcL data sets were performed using PAUP* 4.0b8 (Swofford,
2001) on a Power Macintosh G4. Measures of incongruence like the incon-
gruence length difference (ILD) test have recently been demonstrated not to
be useful as indicators of data partition combinability (e.g., Yoder et al., 2001;
Reeves et al., 2001). Moreover, hypotheses of conflict based on inspection of
trees have been largely supported by subsequent statistical comparisons in
several studies (e.g., Mason-Gamer and Kellogg, 1996). Therefore visual in-
spection of the individual bootstrap consensus trees was used for determining
combinability of the three data sets (Whitten et al., 2000). In case of not
strongly supported (,85% BP) and incongruent patterns between the individ-
ual trees, direct combination was regarded as appropriate. Each included nu-
cleotide position was treated as an independent, unordered, multistate char-
acter 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 MULTREESon (keeping
multiple shortest trees) but holding only 10 trees per replicate to reduce time
spent in swapping on large numbers of suboptimal trees. After these 1000
replicates, we then used the shortest trees found as starting trees for a search
with a tree limit of 15000, which was reached in some cases; this procedure
will often find more trees at this shortest length than were found in the 1000
replicates of random taxon entry. Robustness of clades was estimated using
the bootstrap (Felsenstein, 1985) with 1000 replicates with simple sequence
474 [Vol. 90A
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OURNAL OF
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OTANY
Fig. 1. One of the 3559 most parsimonious trees obtained from the max-
imum parsimony (MP) analysis of the combined data set (26S rDNA, matK,
rbcL) of 41 Meliaceae accessions. Subfamilies and tribes after Pennington
and Styles (1975). Numbers above branches are estimated branch lengths
(ACCTRAN optimization), numbers below branches are bootstrap percent-
ages (1000 replicates). Arrowheads indicate groups not present in the strict
consensus tree.
Fig. 2. Bootstrap consensus tree (MP) of the matK sequence data set of
28 Meliaceae accessions. Subfamilies and tribes after Pennington and Styles
(1975). Numbers are bootstrap percentages (1000 replicates).
addition, TBR branch swapping, and MULTREES on but holding 10 trees per
replicate to reduce time spent on each replicate. As outgroup, representatives
of the closely related families Rutaceae, Sapindaceae, and Simaroubaceae
were used (Gadek et al., 1996; Savolainen et al., 2000b). Patterns of DNA
sequence evolution for each gene was assessed on one of the shortest com-
bined trees. To calculate the number of transitions (ts) and transversions (tv)
and their collective consistency indices (CI) and retention indices (RI), a step
matrix was used to calculate the number of tv by weighting ts to zero. From
tv values, those of the ts were calculated (Whitten et al., 2000).
Bayesian analysis was conducted in MrBayes (Huelsenbeck and Ronquist,
in press) on the three-gene matrix using four Markov chains simultaneously
started from random trees. A simple evolutionary model was used because
more complicated models ran too slowly: a simple two-parameter (transition-
transversion) substitution model (nst 52, rate 5equal and basefreq 5equal).
One million cycles were performed with each model, sampling a tree at every
100 generations. Trees that preceded the stabilization of the likelihood value
(the burn in) were excluded, and the remaining trees were used to construct
a consensus in PAUP (version 4.0; Swofford, 2001).
RESULTS
Parsimony analysis—The mean GC ratios for the sequences
of matK, rbcL, and 26S rDNA were 36%, 44%, and 61%,
respectively. Whereas for the rbcL matrix 170 characters (out
of 1387, i.e., 12.3%) were parsimony informative, 148 char-
acters (out of 893, i.e., 16.6%) were parsimony informative
for matK, and 71 characters (out of 880, i.e., 8.1%) for 26S
rDNA. On the shortest combined tree shown (Fig. 1), evolu-
tion of each gene was assessed. The ts/tv ratio for matK was
1.24, CI and RI for ts were 0.77 and 0.85, for tv 0.62 and
0.83, respectively. The ts/tv ratio for rbcL was 1.36, CI and
RI for ts were 0.68 and 0.84, respectively, and for tv 0.45 and
0.83, respectively. The ts/tv ratio for 26S was 2.88, CI and RI
for ts were 0.45 and 0.53, respectively, and for tv 0.43 and
0.61, respectively.
Inferred phylogenetic trees resulting from five separate anal-
yses are shown in Figs. 1–5: all three regions combined, matK
alone, rbcL alone, 26S rDNA alone, and a combined plastid
analysis. For all but the first, we illustrate only the bootstrap
majority-rule consensus tree because our purpose in analyzing
the data sets separately is simply to demonstrate that no in-
stances of strongly supported and incongruent patterns are pre-
sent so that direct combination is appropriate. Numbers after
genus name in the trees refers to the tribe numbers given in
Pennington and Styles (1975; http://ajbsupp.botany.org/v90/).
Analysis of matK—The aligned matK matrix consisted of
893 bp of which 297 (33%) positions were variable and 148
(17%) of these were potentially parsimony informative. Anal-
ysis produced more than 15000 shortest trees of 460 steps
with a CI of 0.69 and an RI of 0.84. In the matK bootstrap
consensus tree (Fig. 2), Melioideae and tribe Melieae within
this subfamily are supported by 100 bootstrap percentage (BP).
With the exception of Walsura, the remaining representatives
of Melioideae are members of an unresolved clade. Swieten-
March 2003] 475M
UELLNER ET AL
.—M
OLECULAR PHYLOGENETICS OF
M
ELIACEAE
(S
APINDALES
)
Fig. 3. Bootstrap consensus tree (MP) of the rbcL sequence data set of
38 Meliaceae accessions. Subfamilies and tribes after Pennington and Styles
(1975). Numbers are bootstrap percentages (1000 replicates).
Fig. 4. Bootstrap consensus tree (MP) of the 26S rDNA data set of 30
Meliaceae accessions. Subfamilies and tribes after Pennington and Styles
(1975). Numbers are bootstrap percentages (1000 replicates).
ioideae are supported by BP 99. There is no confirmation of
any tribal circumscription, i.e., representatives of the three
tribes within Swietenioideae appear intermixed, but tribal de-
limitations also cannot be refuted from this evidence.
Analysis of rbcL—The aligned rbcL matrix consisted of
1387 bp of which 247 (18%) positions were variable and 170
(12%) of these were potentially parsimony informative. Anal-
ysis produced 612 shortest trees of 474 steps with a CI of 0.58
and an RI of 0.84. In the rbcL bootstrap consensus tree (Fig.
3), Melioideae are monophyletic (BP 94), with BP 100 for
Melieae. The representatives of Guareeae and Aglaieae form
a common clade (BP 76). Members of Turraeeae and Trichi-
lieae are intermixed. Swietenioideae (with Capuronianthus in-
cluded) are monophyletic (BP 67); Swietenieae are paraphy-
letic/polyphyletic, and the two members of Cedreleae (Cedrela
and Toona) are sister (BP 62) to a clade containing some mem-
bers of Swietenieae, Xylocarpeae (Carapa), and Capuronian-
thus. The members of the two monogeneric subfamilies, Quiv-
isianthe and Capuronianthus, are embedded within Melioideae
and Swietenioideae, respectively.
Analysis of 26S—The aligned 26S rDNA matrix consisted
of 880 bp of which 130 (15%) positions were variable and 71
(8%) of these potentially parsimony informative. Analysis pro-
duced 46 shortest trees of 289 steps with a CI of 0.48 and an
RI of 0.64. The bootstrap consensus tree (Fig. 4) shows little
resolution and does not even separate Melioideae from Swie-
tenioideae. It does support the relationship of Carapa (Xylo-
carpeae) to Swietenia and Khaya (the last two Swietenieae; BP
99) seen with rbcL. Melieae are weakly supported (BP 51).
Aglaia is monophyletic (BP 74), and there are several pairs of
genera that are supported: Guarea/Heckeldora (BP 100), Ny-
mania/Trichilia (BP 58), and Turraea/Walsura (BP 93). The
last pair are clearly in conflict with rbcL, which placed Tur-
raea with Calodecaryia (not sequenced for 26S rDNA) and
this pair with Nymania (BP 99 and 71, respectively).
Combined analysis—Except for the above-noted incongru-
ence related to the relationships of Turraea, in none of the
three separate analyses did we observe any other occurrences
of strongly supported incongruence. We noted this potential
case of conflict but proceeded with direct combination in two
phases, one for just the two plastid genes (so that the effect
of adding 26S rDNA to the plastid results could be better
interpreted) and another for all three genes. Analysis of the
combined plastid matrix produced 11564 shortest trees of 934
steps with a CI of 0.63 and an RI of 0.84. In the bootstrap
consensus tree (Fig. 5), Melioideae are monophyletic (BP 98),
with BP 100 for Melieae. The representatives of Guareeae and
Aglaieae form a common clade (BP 60). Members of Tur-
raeeae and Trichilieae are intermixed. Swietenioideae are
monophyletic (BP 84); Swietenieae are paraphyletic/polyphy-
letic, and Capuronianthus and the members of Cedreleae and
Xylocarpeae are nested within them. The members of the two
monogeneric subfamilies, Quivisianthe and Capuronianthus,
are embedded within Melioideae and Swietenioideae, respec-
tively.
476 [Vol. 90A
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Fig. 5. Bootstrap consensus tree (MP) of the combined plastid data set of
41 Meliaceae accessions. Numbers are bootstrap percentages (1000 repli-
cates).
Analysis of the combined three-gene matrix produced 3559
shortest trees of 1251 steps with a CI of 0.57 and an RI of
0.79. In Fig. 1, we illustrate one of the shortest trees; numbers
above branches are estimated numbers of substitution (ACCT-
RAN optimization); numbers below branches are bootstrap
percentages. Groups that are not present in the strict consensus
tree are marked with an arrowhead. This result is more re-
solved than any of the other analyses, including the combined
matK/rbcL; the incongruence of Turraea noted above appears
to have little effect on the combined analysis. Unless otherwise
noted, in the following we will make reference to the results
of the combined three-gene analysis (i.e., joint matrix matK/
rbcL/26S; Fig. 1). Relative to just the plastid results, the ad-
dition of the 26S rDNA data did not alter patterns noticeably
nor were patterns of bootstrap support greatly affected; they
definitely did not increase greatly over those obtained with
combined matK/rbcL, but there appeared to be no negative
effect either.
The combined molecular data support the two main lineages
that correspond to Melioideae and Swietenioideae (BP 100 and
90, respectively). However, Quivisianthe and Capuronianthus
are embedded in Melioideae and Swietenioideae, respectively.
Quivisianthe forms a well-supported (92% BP) clade with Ek-
ebergia, a member of Trichilieae. Capuronianthus forms a
clade (80% support) with Lovoa, a member of Swietenieae.
Concerning tribal delimitation within Melioideae, the data in-
dicate a close relationship between representatives of Aglaieae
and Guareeae, although this does not receive BP .50. The
single tree illustrated shows Guareeae to be paraphyletic to
Aglaieae, but the low levels of sequence divergence observed
prevent us from concluding that there are definite problems
with tribal delimitation. Members of Turraeeae and Trichilieae
are intermixed, but not as strongly as in the individual plastid
results. Due to the low levels of sequence divergence ob-
served, this result cannot be taken as refutation of the mono-
phyly of Turraeeae and Trichilieae. Concerning tribal delimi-
tation in Swietenioideae, the data indicate at least the Swie-
tenieae to be non-monophyletic. The members of Cedreleae
(only two genera) form a weakly supported clade (BP 57).
Only one genus (Carapa) of the two genera of Xylocarpeae
was included.
Bayesian analysis—Bayesian results (tree not shown) are
nearly identical to the parsimony tree (Fig. 1); some weakly
supported groups are differently arranged (e.g., the position of
Synoum), but in neither case does this receive either a high
bootstrap or posterior probability. In some cases, the posterior
probabilities are higher than the bootstrap percentages, but all
clades with high posterior probabilities are also present and
receive at least moderate bootstrap support in the parsimony
analyses.
DISCUSSION
Molecular evolution—Although the percentage of infor-
mative characters is highest in the sequenced matK region se-
quenced here, rbcL provides more informative characters, re-
sulting in the best resolved and supported trees obtained in
this study. Perhaps if we had developed PCR primers that al-
lowed us to sequence the whole matK exon, there would have
been more information in matK, but these primers would have
required a great more time to develop. The results from the
analysis of 26S rDNA were disappointing to us due to the low
number of variable sites and their poor performance. We
would not recommend this region to other researchers for the
purposes of examining infrafamilial relationships.
The appearance of incongruence relating to the position of
Turraea should be noted and investigated further. Although
potentially a case of incongruence, direct combination did not
seem to produce any of the expected signs of strong disagree-
ment, i.e., less resolution and diminished bootstrap percent-
ages. There is no particular reason based on morphology why
Turraea (Turraeeae) should be allied with Walsura (Trichi-
lieae), and in the plastid and combined analyses Turraea
comes out with other Turraeeae. It could be argued that simply
because there are more variable sites in the plastid matrices
that they overwhelm the pattern obtained with 26S rDNA but
in general strongly supported incongruent patterns decrease
support and resolution, so we are uncertain about the best per-
spective from which to view this situation. We note it here so
that future workers will investigate it further.
The ts/tv ratios were as expected for plastid and nuclear
coding regions; for 26S rDNA they were much higher, but
matK had a much less skewed pattern than did rbcL, a fact
noticed by other researchers (Kores et al., 2000; Whitten et
al., 2000). Kores et al. argued that these patterns indicate that
matK might be a pseudogene, but at the least it indicates that
matK has different functional constraints operating on it than
does rbcL. We note here that insertions and deletions in matK
March 2003] 477M
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(S
APINDALES
)
were all in triplets, indicating that matK is now or was recently
functional.
Familial relationships of Meliaceae—As outgroups, we in-
cluded representatives of the putatively closely related families
Rutaceae, Sapindaceae, and Simaroubaceae, but sampling was
too limited here to say much about interfamilial relationships.
Some members of these families have formerly been included
in Meliaceae (De Candolle, 1824, 1878; Hooker, 1862; Radl-
kofer, 1890; Harms, 1896, 1940), but the question of whether
Flindersia and Chloroxylon (usually placed in Rutaceae; e.g.,
Scott et al., 2000) and Ptaeroxylon and Cedrelopsis (previ-
ously assigned to Ptaeroxylaceae: e.g., Gadek et al., 1996;
Chase et al., 1999; but in Rutaceae sensu APG, 1998) should
be included in Meliaceae has been debated for sometime (e.g.,
review in Pennington and Styles, 1975; Chase et al., 1999).
DNA data (Gadek et al., 1996; Chase et al., 1999; Savolainen
et al., 2000b) have shown that the closest relatives of Meli-
aceae are Simaroubaceae and Rutaceae, but Flindersia/Chlo-
roxylon and Ptaeroxylon/Cedrelopsis are excluded from Me-
liaceae (and not included here). Among other families, Meli-
aceae, Rutaceae, Sapindaceae, and Simaroubaceae form the
core group of Sapindales as recognized in Cronquists’s broad
concept of this order (Cronquist, 1968) and confirmed by DNA
studies (Gadek et al., 1996).
Subfamilies—Of the four subfamilies included here, the
two largest, Swietenioideae, and Melioideae have been rec-
ognized in some form or other since 1789 (de Jussieu). Their
rank and circumscription, however, have frequently been de-
bated. Adrien de Jussieu (1830) treated them as families, an
opinion that Harms (1940) supported. Nevertheless, Harms
treated them as subfamilies, together with a third, based on
Cedrela. Apart from the inclusion of Cedrela and the related
taxon Toona, Swietenioideae have remained a remarkably sta-
ble taxon. There has been only serious dispute over the inclu-
sion of the mangrove genus Xylocarpus and the related Car-
apa. Harms (1940) placed them in Melioideae on the basis of
seed characters and Kribs (1930) in Swietenioideae on the ba-
sis of wood anatomy alone. Pennington and Styles (1975),
examining the secondary xylem of a variety of species, pre-
sented evidence that Cedrela (and Toona), Xylocarpus and
Carapa have so much in common with the rest of Swieten-
ioideae that their exclusion was unjustified. Our data (matK,
rbcL, and all combined analyses; Figs. 1, 2, 3, and 5) confirm
that these taxa belong to Swietenioideae. Furthermore the data
indicate a close relationship between Cedrela and Toona,
forming monophyletic clades in the rbcL (Fig. 3) and com-
bined bootstrap analyses (Figs. 1, 3, and 5). Pennington and
Styles (1975) stated furthermore that the secondary xylem of
Swietenioideae is virtually uniform and consistently different
from that of Melioideae. The pollen of most Swietenioideae
and most Melioideae, on the other hand, is so similar that it
confirms the decision to treat them as subfamilies. Both hy-
potheses are strongly confirmed by our study. First of all, the
matK and rbcL trees (Figs. 2 and 3) as well as the combined
trees (Figs. 1 and 5) support recognition of the two main sub-
families. Only the 26S matrix does not provide support for
these two natural groups (Fig. 4). Second, our data (except
26S) support the monophyly of Meliaceae with Melioideae
and Swietenioideae appearing as sister groups and thus are
also compatible with their taxonomic rank as subfamilies
(Figs. 1, 2, 3, and 5).
The study of two little-known Malagasy genera, Capuron-
ianthus and Quivisianthe by Pennington and Styles (1975) lead
to the establishment of two new subfamilies, even though the
authors stated that these genera provided connecting links be-
tween the two larger subfamilies. They were believed to be so
different from each other, as well as from Swietenioideae and
Melioideae, that the establishment of two new subfamilies
seemed to be justified. Our DNA data show that Quivisianthe
and Capuronianthus are embedded in Melioideae and Swie-
tenioideae, respectively, and that this decision therefore cannot
be justified based on phylogenetic grounds (Fig. 3).
Nevertheless it should also be mentioned that this infor-
mation is solely based on the rbcL sequence data because we
were unable to amplify either for matK or 26S rDNA. How-
ever, Pennington and Styles (1975, pp. 445 and 509) stated in
their generic monograph that the floral structure of Quivisian-
the is similar to that of Ekebergia (Trichilieae, tribe 4, of Me-
lioideae). However, the dry, loculicidal capsule containing dry
winged seeds immediately distinguishes Quivisianthe from
members of this tribe as well all other members of Melioideae
and Swietenioideae. The DNA data confirm a close relation-
ship of Quivisianthe to Ekebergia (Trichilieae, BP 93; Fig. 3).
Pennington and Styles (1975, p. 511) suggested a distant
relationship of Capuronianthus with Carapa and Xylocarpus
(Swietenioideae), with which it shares both the partial septif-
ragal dehiscence of the fruit (both genera) and a seed with
corky sarcotesta (only Xylocarpus). In addition to these, Car-
apa and Capuronianthus have the same chromosome number
(2n558). However, in all other characters of subfamilial im-
portance Capuronianthus differs from these genera. Moreover,
Pennington and Styles (1975) found that in some floral char-
acters, Capuronianthus resembles closely genera in Trichilieae
(Melioideae). The loculi containing only two fully developed
ovules and the capitate style-head are characteristic of many
members of the Melioideae, although Pennington and Styles
(1975) pointed out that an additional vestigial ovule in the
loculus occurs rarely in the latter subfamily. In contrast, loculi
with three or more ovules are typical for members of Swie-
tenioideae. In its secondary xylem Capuronianthus is inter-
mediate between Swietenioideae and Melioideae. Pennington
and Styles (1975) thus concluded that the genus is intermediate
between the two subfamilies but nevertheless quite distinct
from both, thus justifying the establishment of the subfamily
Capuronianthoideae. In the molecular trees (Figs. 1, 3, and 5)
Carapa (as well as Khaya and Swietenia) are the closest rel-
atives of Capuronianthus after Lovoa. Bootstrap support for
the clade formed by Capuronianthus and Lovoa is 83% (Fig.
3), and this group is sister to a clade formed by the genera
Carapa,Khaya, and Swietenia. Due to the fact that Capuron-
ianthus is positioned within Swietenioideae, this genus—like
Quivisianthe—should not be treated as a subfamily.
Tribes—De Candolle in his Prodromus (1824) was the first
to attempt to divide the family into tribes, which he based
primarily on the number and structure of seeds. A still more
detailed account was published in 1830 by Adrien de Jussieu.
De Jussieu’s classification was an improvement because it was
based on a larger number of characters than any previous clas-
sification. Hooker in Genera Plantarum (Hooker, 1862) fol-
lowed de Candolle (1824) but differed from de Jussieu (1830)
in uniting Cedrelaceae and Meliaceae. All subsequent authors
have done the same.
Since the publication of Bentham and Hooker’s Genera
478 [Vol. 90A
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Plantarum, four comprehensive classifications of the family
have been published: one by Casimir de Candolle (1878), two
by Harms (1896, 1940), and one based on the anatomy of the
secondary xylem by Kribs (1930). As has been already stated,
the most authoritative work on generic and tribal delimitation
in Meliaceae at present is the generic monograph by Penning-
ton and Styles (1975), although since then a considerable num-
ber of new insights and reevaluations with respect to morpho-
logical characters has led to slight modifications (e.g., Mab-
berley, 1979; Cheek, 1989, 1990a, b, 1992, 1996; Cheek and
Rakotozafy, 1991). Some genera and most tribes can only be
diagnosed by using a combination of several characters. Thus,
members of one tribe cannot be distinguished from all other
Meliaceae on the basis of single diagnostic characters. Most
character states typical of one tribe may have at least a few
exceptions in this tribe and also occur at least occasionally in
other tribes (Pennington and Styles, 1975). The DNA data col-
lected so far in this study only support the historically stable
Melieae (Melioideae; 100% BP in the matK, rbcL, combined
plastid and three-gene trees respectively; Figs. 1, 2, 3, and 5).
This tribe comprises the first two species (Melia azedarach,
Azadirachta indica) recognized in the family in the first edition
of Species Plantarum (1753). All members of this tribe also
have a unique six base-pair insertion in matK (59-TTAAGT-
39) at site 137–142 (relative to Melia azedarach, GenBank no.
AY128193) . There is also phytochemical evidence for the
special position of Melieae (compare Taylor in Pennington et
al., 1981; Mulholland et al., 1998). In spite of the thorough
investigation of Melia and Azadirachta in recent years, gla-
bretal-type compounds (triterpenoids) have not been found in
these genera in contrast to all other members of Melioideae
investigated so far (Aglaia,Guarea,Owenia,Turraea, and Dy-
soxylum; Mulholland et al., 1998). For all other tribes, there
is not yet enough evidence to evaluate their monophyly. Nev-
ertheless, the preliminary data indicate a close relationship be-
tween Aglaieae and Guareeae (Melioideae) and a possibly
monophyletic Cedreleae (Swietenioideae; Figs. 1, 3, and 5).
Non-monophyletic are at least Trichilieae (Melioideae) and
Swietenieae (Swietenioideae; Figs. 1, 3, and 5). To reach a
robust and well-resolved phylogenetic appreciation of Meli-
aceae, sampling of additional taxa and the collection of many
more data will be necessary.
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