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(American Journal of Botany. 2001;88:697-705.)
© 2001 Botanical Society of America, Inc.

Molecular phylogenetics of Acacia (Fabaceae: Mimosoideae) based on the chloroplast MATK coding sequence and flanking TRNK intron spacer regions1

Joseph T. Miller2 and Randall J. Bayer

Centre for Plant Biodiversity Research, CSIRO Plant Industry, GPO Box 1600, Canberra, Australia 2601

Received for publication January 20, 2000. Accepted for publication June 15, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The tribe Acacieae (Fabaceae: Mimosoideae) contains two genera, the monotypic African Faidherbia and the pantropical Acacia, which comprise about 1200 species with over 950 confined to Australia. As currently recognized, the genus Acacia is subdivided into three subgenera: subg. Acacia, subg. Aculeiferum, and the predominantly Australian subg. Phyllodineae. Morphological studies have suggested the tribe Acacieae and genus Acacia are artificial and have a close affinity to the tribe Ingeae. Based on available data there is no consensus on whether Acacia should be subdivided. Sequence analysis of the chloroplast trnK intron, including the matK coding region and flanking noncoding regions, indicate that neither the tribe Acacieae nor the genus Acacia are monophyletic. Two subgenera are monophyletic; section Filicinae of subgenus Aculeiferum does not group with taxa of the subgenus. Section Filicinae, eight Ingeae genera, and Faidherbia form a weakly supported paraphyletic grade with respect to subg. Phyllodineae. Acacia subg. Aculeiferum (s. s.) is sister to the grade. These data suggest that characters currently used to differentiate taxa at the tribal, generic, and subgeneric levels are polymorphic and homoplasious in cladistic analyses.

Key Words: Acacia • chloroplast DNA • Ingeae • matK • phylogeny


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Bentham (1842) described the tribe Acacieae Benth. as one of three tribes comprising the subfamily Mimosoideae and included within it many genera that are today classified in tribe Ingeae Benth. Later Bentham (1875) restricted his definition of tribe Acacieae to include only a single genus Acacia Mill. As currently defined, tribe Acacieae contains only two taxa, the large cosmopolitan genus Acacia and the monotypic African genus Faidherbia A. Chev. (Vassal, 1972, 1981 ).

The main character that distinguishes the Acacieae from the Ingeae, free filaments of the stamens while the Ingeae has united filaments, is not maintained in all taxa with some having filaments shortly united at base (Vassal, 1981 ). Other characters shared between the tribes are: numerous stamens and eight polyads per anther (Chappill and Maslin, 1995 ) and the close relationship of the Ingeae and Acacieae has been noted (Guinet, 1981 ; Vassal, 1981 ). The relationship of Faidherbia is troublesome as it has stamens that are shortly united at base and has pollen similar to some taxa of the Ingeae, but was placed in the Acacieae (Guinet, 1981 ). The tribe Mimoseae Bronn shares the character state of free stamens with the Acacieae, but the Mimoseae has as many or twice as many stamens as petals while the Acacieae has numerous stamens (Vassal, 1981 ). Guinet (1990) noted pollen structural symmetry shared among some Mimoseae and Acacia subg. Acacia. These conflicting character states make a classification, based solely on morphological characters, difficult.

Within Acacia, Bentham (1875) recognized six series, but recent authors have amalgamated these into three major groups (Table 1) either at the generic or subgeneric level (Vassal, 1972 ; Pedley, 1986 ; Maslin and Stirton, 1997 ). Subgenus Acacia and subg. Aculeiferum Vassal, with over 120 and 180 species, respectively, are pantropical while subg. Phyllodineae (DC.) Seringe, with over 950 species, is largely confined to Australia (Ross, 1981 ; Maslin and Stirton, 1997 ).


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  		Table 1. Classification of the Acacieae as adopted in present paper. This is a synaptic scheme based on Vassal (1972) and Pedley (1978, 1986). Under subgenus, the parenthetical generic names are those adopted by Pedley (1986)

 
Subgenus Acacia has bipinnate leaves, stipular spines, and colporate pollen with a smooth exine with columellae, whereas subgenus Aculeiferum has bipinnate leaves, no stipular spines, but may have two to three prickles near the stipules, and porate pollen with a smooth exine but without columellae (Vassal, 1981 ). Subgenus Phyllodineae is the more diverse and variable of the subgenera. Most species have leaves reduced to vertically flattened phyllodes in a diverse range of sizes and shapes, but others have bipinnately compound leaves. They do not have prickles, but can be spinescent and have extraporate or porate pollen with the exine reticulate without columellae (Vassal, 1981 ).

Sections among the phyllodinous taxa have been derived based on phyllode nervature and inflorescence structure (Pedley, 1978 ). While the sections may not be considered as natural groups (Pedley, 1986 ; Brain and Maslin, 1996 ; Chappill and Maslin, 1995 ) they form a useful framework for investigation (Table 1). Section Phyllodineae contains species with one-nerved phyllodes while sects. Juliflorae (flowers in spikes) and Plurinerves (Benth.) Maiden & Betche (flowers in heads) taxa have multinerved phyllodes. Within these plurinerved taxa differences can be noted between microneurous phyllodes (numerous, fine longitudinal nerves) and oligoneurous phyllodes (few, distant longitudinal nerves; Maslin and Stirton, 1997 ).

There is growing agreement among researchers that the genus needs to be divided, but there is uncertainty regarding the number of terminal taxa involved, their interrelationships, and their taxonomic rank (Maslin and Stirton, 1997 ). In particular, there is disagreement on interrelationships of the three subgenera. Pedley (1986) hypothesized that subg. Phyllodineae arose from within subg. Aculeiferum and that subg. Aculeiferum and subg. Acacia had separate origins from within the tribe Ingeae. A recent morphological cladistic analysis also showed a sister relationship between subg. Aculeiferum to subg. Phyllodineae (Chappill and Maslin, 1995 ). These two genera were nested within the Ingeae, but separate from subg. Acacia that was also nested within the Ingeae. Alternatively, a cladistic analysis of floral development morphology suggests a close relationship of subgenera Acacia and Aculeiferum nested in the Ingeae separate from subg. Phyllodineae (Grimes, 1999 ).

The character states inconsistencies among the tribes, character polymorphisms within tribes and genera, and disagreement among published analyses indicate that a reevaluation of the tribes based upon an independent, data set is necessary. The aim of this study was to test the monophyly of tribe Acacieae and of the genus Acacia and to investigate phylogenetic relationships among the three subgenera within Acacia using chloroplast DNA sequences. To accomplish this goal, the cpDNA intron of the transfer RNA gene for lysine (trnK) was sequenced. This region includes the maturase encoding gene (matK) as well as flanking noncoding regions. The entire intron is ~2300 bp with the coding region of matK being ~1500 bp. The matK evolves two- to threefold faster than rbcL (Johnson and Soltis, 1994 ; Plunkett, Soltis, and Soltis, 1997 ), and this sequence has been used mainly at the infrageneric level, where a faster evolving chloroplast gene than rbcL is desired.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
A recent generic and infrageneric classification outlined "a list of critical species on which to build a comparative data set" (Maslin and Stirton, 1997 ). This list describes morphological groups within each subgenus that could be used to systematically sample the large number of species in the genus. The Acacieae ingroup sampling of the present study was based on these morphological groups. Species were sampled evenly from all three subgenera of Acacia (Table 2) and the monotypic Faidherbia albida was also included.


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  		Table 2. Sources of DNA used in this study

 
Eight genera from the Ingeae and a single genus, Mimosa, of the Mimoseae were included as outgroup taxa. Preliminary analysis indicated that the Ingeae taxa were ingroup taxa as they nested within the Acacieae in all analyses. In subsequent analyses, Mimosa tenuiflora was used as the outgroup. The selection of these outgroup taxa was based on morphological evidence (Chappill and Maslin, 1995 ; Grimes, 1999 ).

Seeds were acquired from various seed banks (Table 2), scarified, placed into a petri dish with Whatman paper, and left to germinate at 25°C with 12 h of light per day. The first true leaf was detached and pulverized in liquid nitrogen. DNA was extracted using a Plant DNAZOL Reagent kit (GIBCOBRL, Grand Island, New York, USA). Initial DNA amplification used the trnK-3914 and trnK-2R primers made from Saxifragaceae (Johnson and Soltis, 1994 ). An Acacia specific primer (Fig. 1) was created internal to trnK-2R and was used in all subsequent polymerase chain reaction (PCR) reactions. The trnK intron region was amplified via the PCR using Taq DNA polymerase (Perkin-Elmer Applied Biosystems, Norwalk, Connecticut, USA). The PCR reaction mixture consisted of 5 µL of 20x reaction buffer, 6 µL of 25 mmol/L magnesium chloride solution, 16 µL of a 1.25 mmol/L dNTP solution in equimolar ratio, 25 pmol of each primer, 10–50 ng of template DNA, and 1.0 unit of polymerase in a total volume of 100 µL. The PCR samples were heated to 94°C for 3 min prior to the addition of DNA polymerase to denature unwanted proteases and nucleases. The double-stranded PCR products were produced via 30 cycles of denaturation (94°C for 1 min), primer annealing (48°C for 1 min), and extension (72°C for 2 min). A 7-min final extension cycle at 72°C followed the 30th cycle to ensure the completion of all novel strands.



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  		Fig. 1. Primers used in this study. (A) Structure of the chloroplast trnK intron including the matK coding sequence. Arrows above figure represent primers used for PCR, and arrows below indicate sequencing primers. Primer trnK-3914 was used for both PCR and sequencing reactions. Primer positions are relative to the start codon of A. penninervis. (B) Sequences of the primers used in this study. Primers trnK 3914 and trnK-2R are from Johnson and Soltis (1994)

 
Double-stranded PCR products were cleaned with the QIAquick PCR kit (QIAgen, Hilden, Germany) and were sequenced using the dideoxy chain termination method with the use of the Big Dye Terminator RR Kit® and an ABI automated sequencer (Perkin-Elmer Applied Biosystems, Norwalk, Connecticut, USA) at CSIRO, Plant Industry. An annealing temperature of 57°C was used for sequencing reactions. Initial sequences were generated with primer trnK-3914. Four Acacia specific internal sequencing primers (Fig. 1) were designed and made, based on initial sequence data. The cycle sequencing protocol followed manufacturer's instructions.

Chromatographic traces and contiguous alignments were edited using SequencherTM 3.0 (Gene Codes Corporation, Ann Arbor, Michigan, USA). All sequences were deposited in GenBank (Table 2). The coding region was determined by comparison to Rosa persica (Genbank number GBAN-AB011974). Sequences were aligned manually with minimal gaps and base substitutions. Indels were scored as separate characters. The matK coding region and the flanking spacer region were analyzed separately and the entire sequence analyzed together. The data were analyzed with all characters unweighted. A second analysis double weighted transversions over transitions. For this second analysis, the matK coding region was weighted as follows: the second-codon position was weighted twice the third position and all noncoding sequence, while the first position was weighted twice the second position.

Maximum parsimony analyses were performed on the aligned sequences using the heuristic search option (excluding uninformative characters) in PAUP 4.02 (Swofford, 1999 ). A four-step search method for multiple islands was performed using 10 000 random replicates (Olmstead and Palmer, 1994 ). Support for internal branches was evaluated by using the fast bootstrap method with 10 000 replicates (Felsenstein, 1985 ).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Sequence characteristics
The aligned length of the sequenced portion of the trnK intron was 2448 bp with 1557 bp forming the matK coding region and an additional 891 nucleotides sequenced in the flanking intron region (Table 3). The intron sequence contained 192 informative base substitutions and 11 indels. Most of the indels (8/11) were in the 5' noncoding region. The mean divergence among taxa was greatest in the small 50-bp section 3' to the matK coding region. The matK coding region codes from 502 to 515 amino acids. The highest divergence (7.4%) in the 5' region was between A. penninervis and A. schaffneri, while the divergence between the outgroup Mimosa and the Ingeae genus Pararchidendron was the highest for the matK coding region (4.0%).


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  		Table 3. Nucleotide character statistics for the trnK/matK region. na = not applicable

 
Of the 11 indels, four were autapomorphic for A. boliviana of Acacia subg. Aculeiferum sect. Filicinae. Subgenus Acacia has two clade specific indels and a homoplasious indel was shared with subg. Aculeiferum sect. Filicinae. Indels also supported clades within subg. Acacia and subg. Aculeiferum sect. Aculeiferum.

Topology of the major clades
Topologies of the cladograms derived from the spacer and matK regions were congruent with slightly better resolution on the data from the matK coding region. Maximum parsimony analysis of the entire unweighted data set found 2236 trees of 398 steps with a CI of 0.62 and an RI of 0.83 (Farris, 1989 ). The topology of the strict consensus tree (Fig. 2) has four basic components: (1) a clade (A) of Acacia subg. Acacia, (2) a clade (B) of Acacia subg. Aculeiferum sects. Aculeiferum and Monacanthea, (3) a grade (C) including Faidherbia albida, all Ingeae genera included in the study, and A. boliviana of Acacia subg. Aculeiferum sect. Filicinae, and (4) a clade (D) comprising Acacia subg. Phyllodineae.



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  		Fig. 2. One of 2236 most parsimonious trees. A dashed line indicates branches that collapse in the strict consensus tree. Numbers below the lines indicate branch length. Numbers above some lines indicate bootstrap support value. Indels are indicated by boxes. A = Acacia subg. Acacia, B = Acacia subg. Aculeiferum, C = Faidherbia/Acacia subg. Aculeiferum sect. Filicineae/Ingeae, D = Acacia subg. Phyllodineae. All taxa of clade D are Australian.

 
Constraint analyses were conducted to test the monophyly of Acacieae and Acacia. When the tribes Acacieae and Ingeae were constrained as monophyletic and the analysis repeated, an additional 12 steps were added to the most parsimonious tree. An additional ten steps were added to the shortest tree when Acacia (without F. albida) was constrained as monophyletic. Neighbor-joining analysis also placed the Ingeae within the polyphyletic Acacieae.

The trees derived from the weighted analysis (not shown) differed from the unweighted analysis by switching the positions of the subg. Acacia and subg. Aculeiferum (s. s.) clades. The sister position of subg. Acacia to the Filicinae/Faidherbia/Ingeae was weakly supported by a bootstrap value of <50%. The other major difference was that the weighted analysis placed F. albida within the Ingeae.

Topology of clades with Acacia
Subgenus Acacia
The basal portion of the strict consensus tree (Fig. 2) contains two clades, (1) Acacia subg. Acacia (Fig. 2A) and (2) the rest of the taxa studied (Fig. 2B, C, D). This indicates a significant divergence of Acacia subg. Acacia from other ingroup taxa. Acacia subg. Acacia formed a well-supported clade with 21 synapomorphies (SYN = 21) and 100% bootstrap support (BV = 100%). Two synapomorphic indels and a homoplasious indel, which is shared with Acacia subg. Aculeiferum sect. Filicinae, support the clade (Fig. 2).

One clade consists of the African species A. seyal, A. karroo, A. nilotica, and A. tortilis with the Australian species A. bidwillii (SYN = 7; BV = 99%). A second clade (SYN = 2; BV = 84%) consists of two New World groups, the A. farnesiana group (A. caven and A. schaffneri; SYN = 9; BV = 100) and the A. macracantha group (A. cochliacantha and A. pennatula; SYN = 7; BV = 100%). The third clade is the New World A. constricta group (A. constricta and A. schottii; SYN = 3; BV = 96%). The A. farnesiana group and the A. constricta group were defined by Clarke, Seigler, and Ebinger (1989, 1990 , respectively).

Subgenus Aculeiferum
Acacia subg. Aculeiferum forms a monophyletic clade when A. boliviana of sect. Filicinae is excluded (Fig. 2B). This species appears as part of a basal grade (C) to subg. Phyllodineae. The rest of the subgenus is supported as monophyletic (SYN = 3; BV = 81%) and is sister to the Ingeae/Filicinae/Faidherbia grade. All branches within the clade have bootstrap support of >88%.

Clade B comprises two clades that correlate with sect. Aculeiferum (SYN = 15; BV = 100%) and Monacanthea (SYN = 13; BV = 100%). Section Aculeiferum is confined to Africa and Asia, whereas sect. Monacanthea is pantropical. Two clades appear within the sect. Monacanthea clade, one (SYN = 7; BV = 100%) is the A. berlandieri group (A. berlandieri and A. wrightii; sensu Maslin and Stirton, 1997 ). The other clade (SYN = 6; BV = 100%) consists of A. glomerosa of the A. glomerosa group and A. bonariensis, which has affinities to the A. riparia group (Maslin and Stirton, 1997 ).

The sect. Aculeiferum clade is further supported by one indel. In the basal position of this clade is A. catechu with paired prickles; a more derived element of the clade (A. senegal) with prickles in threes (Ross, 1979 ) grouped with the Indian taxon A. modesta (SYN = 4; BV = 88%).

Faidherbia, Ingeae and sect. Filicinae
A grade consisting of Faidherbia albida, A. boliviana of Acacia subg. Aculeiferum sect. Filicinae and the eight Ingeae genera (Fig. 2C) is basal to a clade containing Acacia subg. Phyllodineae. These taxa are from Australia, the New World, and Africa. This grade is weakly supported with only two branches having a bootstrap value >50%. The sister position of Faidherbia albida is supported by a 69% bootstrap value and seven synapomorphies. The Ingeae are not monophyletic. An Australian species of Pararchidendron is basal to a trichotomy. The trichotomy consists of (1) six of the eight Ingeae genera, (2) the Australian species Cathormion umbellatum, and (3) a monophyletic Acacia subg. Phyllodineae clade. Relationships within these Ingeae taxa are not well supported, with only high support for the sister relationship of Ebenopsis and Havardia (SYN = 7; BV = 99%).

Subgenus Phyllodineae
Acacia subg. Phyllodineae formed a monophyletic clade (Fig. 2D). The support for this clade is lower than for clades representing the other subgenera (SYN = 3; BV = 56%). Because this subgenus contains over 950 species (Maslin and Stirton, 1997 ), the 18 taxa sampled here cannot represent the complexity of the subgenus. The four largest of the seven sections (Tables 1 and 2) recognized by Pedley (1978) are represented, including uninerved and plurinerved phyllodinous species as well as species with bipinnately compound leaves.

The clade is unresolved with five lineages. (1) Two uninerved taxa with racemose inflorescences referable to section Phyllodineae, Acacia ligulata of the A. bivenosa group (Chapman and Maslin, 1992 ) and A. myrtifolia of the A. myrtifolia group (Maslin, 1995a ), group together with four synapomorphies. (2) The uninerved nonracemose A. siculiformis does not group with any other taxon. (3) Acacia lineata, a uninerved nonracemose species of sect. Phyllodineae, occurs with the plurinerved oligoneurous A. melanoxylon of sect. Plurinerves. (4) A clade of five plurinerved species of sects. Plurinerves and Juliflorae are joined with the uninerved nonracemose A. rossei, sect. Phyllodineae, basal (SYN = 11). Two closely related species pairs occur within the clade. Acacia monticola and A. lysiphloia are supported as sister species (SYN = 6; BV = 78%). Also A. nuperrima and A. translucens are sisters supported by two synapomorphies. These two groups were also suggested by Maslin and Stirton (1997) . (5) The final clade of the trichotomy is composed of four species with uninerved phyllodes and racemose inflorescences of sect. Phyllodineae and two bipinnate leaved species of sect. Botrycephalae, A. elata and A. parramattensis. The basal relationship of A. fasciculifera is supported by a single synapomorphy. Better supported (SYN = 6; BV = 78%) is the relationship of the uninerved racemose species of the A. microbotrya group (Maslin, 1995b ) with two species of sect. Botrycephalae. The two bipinnate sect. Botrycephalae species do not form a clade, but individually group with uninerved species A. bancrofiatum and A. penninervis. The two other species of the A. microbotrya group (A. microbotrya [SYN = 3; BV = 87%] and A. notabilis) are basal taxa to the clade that contains the bipinnate Botrycephalae species.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
This study begins a series of investigations on the molecular systematics of the genus Acacia. The primary objective of the present work was to test the monophyly of Acacia and tribe Acacieae using DNA sequence data from the matK\trnK chloroplast region. The evidence presented here clearly shows that the genus and tribe are polyphyletic and agree with data from the nuclear Histone H3 data from the same taxa (Miller and Bayer, 2000 ). These results show eight genera of the tribe Ingeae embedded within Acacia and Acacieae.

Based on previous work (Pedley, 1986 ; Chappill and Maslin, 1995 ; Grimes, 1999 ) this study sampled several genera of Ingeae to test the monophyly of Acacia and Acacieae. While this sampling is sufficient to negate the monophyly of the genus and tribe, it is not sufficient to distinguish the interrelationship between the two tribes or their relationship to tribe Mimoseae.

The placement of the Ingeae genera within the Acacia clade demonstrates the polyphyly of the latter. The polyphyly of Acacieae concurs with the original description of the tribe by Bentham (1842) , who described it as containing taxa presently placed in both Acacieae and Ingeae. This result is not surprising since there is not a single morphological character that can separate the tribes (Chappill and Maslin, 1997). The Acacieae has been distinguished from the Ingeae by having free filaments of the stamens while the Ingeae has united filaments, however Faidherbia albida and a few species from all three Acacia subgenera have shortly united filaments (Vassal, 1981 ) and some species of Ingeae have filaments that are almost free (Guinet, 1990 ). Thus without the filament character there are no macromorphological characters that separate the Ingeae and Acacieae, but rather suites of character state changes are needed to separate the tribes. Other characters shared between the tribes are numerous stamens and eight polyads per anther (Chappill and Maslin, 1995 ).

These data suggest an amalgamation of the Ingeae and Acacieae is needed. However, given that morphological characters previously used to discriminate the tribes are sometimes polymorphic within tribes, further molecular and morphological analyses of the entire subfamily Mimosoideae, especially the tribe Mimoseae, are needed before major realignment can be confidently determined. The Ingeae and Acacieae are thought to be derived from a paraphyletic Mimoseae (Pohill, Raven, and Stirton, 1981 ), and analyses of the three tribes together will shed light on the phylogeny and morphological character state changes in the Mimosoideae.

As noted above, Faidherbia albida contains a unique suite of filament and gland characters which are found individually in Acacia species (Ross, 1979 ). This has led to its placement in the Ingeae and Acacieae, and the present analysis is equivocal, but places it as a unique lineage within the Acacieae/Ingeae. The matK/trnK intron sequence data weakly ally Faidherbia as basal to Acacia sect. Filicinae and tribe Ingeae. These results show no clear sister relationship of Faidherbia to any particular Acacieae/Ingeae taxa and suggest generic status for Faidherbia is appropriate. Increased sampling of Ingeae taxa are needed to address the phylogenetic position of Faidherbia.

Bentham (1875) originally proposed six series within Acacia with most authors now regarding them as comprising three subgenera or genera (Vassal, 1972 ; Pedley, 1986 ; Maslin and Stirton, 1997 ). The distinction among the three subgenera is supported by the present molecular data except that Acacia subg. Aculeiferum sect. Filicinae is a separate lineage from other taxa of the subgenera. This supports Bentham's series Filicinae separate from series Vulgares. Series Vulgares has been placed into Vassal's subg. Aculeiferum (Vassal, 1972 ; Pedley, 1986 , i.e., Senegalia). Guinet and Vassal (1978) placed sect. Filicinae within subgenus Aculeiferum based primarily on pollen characters, but noted that the section retains several ancestral morphological character states of the genus.

A high degree of resolution and branch support found within subg. Aculeiferum and Acacia clades suggests that the trnK/matK region is well suited to phylogenetic analysis in these group. Two classes of subg. Acacia (Fig. 2) correlate to the New World Pluriseriae, having seeds in two or three series within the fruit, and the African Uniseriae, having seeds in one series in the fruit (Vassal, 1972 ). Also within subg. Aculeiferum two clades are found that correlate to the New World taxa of sect. Monacanthea and the Africa/Asia taxa of sect. Aculeiferum.

Many hypotheses based on morphological characters have been put forward on the relationships among the three Acacia subgenera. Disagreement of relationships by workers suggests that the morphological characters used are not sufficient to delimit phylogeny of the group. Several studies have suggested a closer affinity of subg. Phyllodineae to subg. Aculeiferum than to subg. Acacia. This relationship is found in the present study with subg. Aculeiferum sister to the clade containing the grade C and the subg. Phyllodineae (Fig. 2). A cladistic analysis of morphological data (Chappill and Maslin, 1995 ; with terminal taxa as genera, subgenera, or sections) indicated a sister relationship of the subg. Aculeiferum (including sect. Filicinae) to subg. Phyllodineae with these two subgenera placed basal to the Ingeae. This relationship, of subg. Phyllodineae and Aculeiferum, was also suggested by Pedley (1986) who proposed the origin of the subg. Phyllodineae from subg. Aculeiferum, which in turn arose from the Ingeae.

The matK/trnK intron data indicate that subgenus Acacia is distinct from the other two subgenera. This is in agreement with Pedley (1986) , while the morphological cladistic analysis shows subg. Acacia as distinct from the other subgenera and is placed within the Ingeae, sister to Calliandra and close to Pithecellobium (Chappill and Maslin, 1995 ). These two genera have not been sampled in the present study but clearly subg. Acacia is not closely related to other Ingeae genera. An alternative result based on inflorescence morphology (Grimes, 199 9) and cpRFLP data (Bukhari, Koivu, and Tigerstedt, 1999 ) did find a close relationship of subgenus Acacia to other Acacieae taxa. An inflorescence morphology development study (Grimes, 199 9) placed subg. Acacia with Faidherbia and subg. Aculeiferum (excluding sect. Filicinae) in a clade that in turn was nested within the New World Pithecellobium complex (Havardia and Ebenopsis in the present study) of the Ingeae. Likewise the chloroplast restriction fragment-length polymorphism study (Bukhari, Koivu, and Tigerstedt, 1999 ) indicates a sister relationship between subg. Acacia and subg. Phyllodineae (Heterophyllum); however this study only included Acacieae species so that no inferences on the monophyly of the genus can be concluded.

Difference among studies may be due to the questions asked in the research. Chappill and Maslin (1995) focused on the Acacieae with a few Ingeae outgroup taxa, and Grimes (1999) focused on the Ingeae with a few Acacieae outgroup taxa. Thorough sampling of the Acacieae, Ingeae, and Mimoseae is necessary to address these questions.

Three of Bentham's series (Pulchellae, Botrycephalae, and Phyllodineae) have been amalgamated into the Phyllodineae (Vassal, 1972 ). The separation of sect. Botrycephalae (a group of Australian bipinnate species) from sect. Phyllodineae is not supported by the matK/trnK intron data as it would leave sect. Phyllodineae paraphyletic.

The chloroplast data presented here indicate that the Botrycephalae is derived from the sect. Phyllodineae. These results suggests that the bipinnate condition of the Botrycephalae is a reversal to the ancestral character state of the Mimosoideae (Pedley, 1986 ). The origin of the Botrycephalae from particular groups of uninerved racemose species, A. microbotrya and its allies, is supported by other studies that considered morphology (Chappill and Maslin, 1995 ), phytochemicals (Tindale and Roux, 1969, 1974 ), and serological data (Brain and Maslin, 1996 ). A more detailed analysis of relationships within subg. Phyllodineae is in progress by the present authors.

The strict consensus tree (Fig. 2) contains a grade consisting of Acacia subg. Aculeiferum sect. Filicinae/Faidherbia/Ingeae (Fig. 2C) that is nested within Acacia. Within this grade there are few synapomorphies and many terminal autapomorphies (8–15), but, nonetheless, only one branch collapses in the strict consensus tree (Fig. 2). This grade may be due to limited sampling of the Ingeae taxa (Bininda-Emonds, Bryant, and Russell, 1998 ; Hillis, 1998 ) since only 8 of ~30 genera of tribe Ingeae have been sampled in the present investigation.

It is possible that long-branch attraction may have artificially affected the grouping of Faidherbia/sect. Filicinae/Ingeae/subg. Phyllodineae. A subset analysis using 13 placeholders from the major clades was conducted to test this possibility. The essential topology of the strict consensus tree was recovered in this analysis (results not shown); however, Acacia subg. Aculeiferum sect. Filicinae and Pararchidendron were included in the unresolved polytomy that previously contained only Cathormion, other Ingeae genera, and subg. Phyllodineae. Faidherbia remained sister to this polytomy. Consequently, caution must be used when interpreting this portion of the cladogram. Sampling of more taxa and additional sequence data may help resolve this issue.

In conclusion, DNA sequence data from the chloroplast matK/trnK region clearly indicates that the Acacia and tribe Acacieae are polyphyletic. Subgenera Acacia and Phyllodineae are monophyletic, while subg. Aculeiferum is polyphyletic. The tribe Ingeae is nested within the Acacieae. The nomenclatural changes necessitated by creation of at least two new genera from Acacia are enormous as they potentially affect >1000 species worldwide. The 39 species of Acacia sampled, representing <5% of the genus, is clearly not enough to elucidate interrelationships within subgenera and sections. More sequence data and increased sampling will be needed before more specific subgeneric interrelationships can be elucidated.

These data suggest a large-scale molecular and morphological reinvestigation of the Ingeae, Mimoseae, and Acacieae is necessary to determine phylogenetic relationships. These data and morphological studies (Chappill and Maslin, 1995 ; Grimes, 1999 ) suggest that characters currently used to differentiate taxa are polymorphic within tribes and genera and homoplasious in cladistic analyses. Pollen and anther characters are not sufficient at the tribal level, and within subg. Phyllodineae leaf type, venation, and inflorescence structure are not always synapomorphic characters. Continued use of molecular methods is critical to determine phylogenetic relationships and to understand character evolution in the Mimosoideae.


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  		Table 2. Continued

 

   FOOTNOTES
 
1 The authors thank Laurie Adams, Les Pedley, Bruce Maslin, The Australian National Botanic Garden, Australian Tree Seed Centre, Oxford Forestry Institute, and the Boyce Thompson Desert Legume Program for supplying material used in this study and Laurie Adams, Tony Brown, Curt Brubaker, Rogier deKok, Don Les, Bruce Maslin, and two anonymous reviewers for suggested improvements to our manuscript. Back

2 Author for correspondence. Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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