Supplementary Discussion
The genus Acacia comprises approximately 1350 species1 and belongs together with the
monotypic African genus Faidherbia Chev.2,3 to the tribe Acacieae, which forms part of the
subfamily Mimosoideae (Fabaceae). Acacia currently is divided into three subgenera: subg.
Acacia (pantropical, ca. 160 species), subg. Aculeiferum Vassal (pantropical, ca. 235 species)
and subg. Phyllodineae (DC) Seringe (syn. subg. Heterophyllum Vassal; ca. 960 species,
predominantly confined to Australia). Pedley4 proposed generic rank for these subgenera,
namely Acacia, Senegalia Raf. and Racosperma C. Martius, but this concept of narrowly
circumscribed genera was not widely accepted.
A number of studies have analyzed morphological, anatomical, palynological and
phytochemical characters as well as molecular markers to ascertain the taxonomic status of
Acacia and to elucidate the phylogenetic relationships within the genus and the tribe
Acacieae1-13. Restriction fragment analyses of chloroplast DNA13-15 and sequence information
from different chloroplast loci and nuclear rDNA (ITS, 5S rDNA) have contributed much to a
better understanding of the phylogenetic relationships within the genus Acacia and the tribes
Acacieae, Mimoseae and Ingeae, respectively9,12,16-18. Molecular studies in general indicate
that the tribes Acacieae and Ingeae as well as the genus Acacia are not monophyletic, whereas
Acacia subg. Acacia and subg. Phyllodineae each appear monophyletic12,13,15-18. However, the
topologies of the cladograms differ remarkably in the relationships of the three subgenera of
Acacia, Faidherbia, and the tribe Ingeae. A recent sequence analysis17 included a broad taxon
sampling of subfamily Mimosoideae and reported a close relationship among Acacia subg.
Acacia and certain members of tribe Mimoseae, while subg. Aculeiferum, the tribe Ingeae,
and Acacia subg. Phyllodineae form a derived clade. Additionally, cladistic analysis of
morphological, anatomical, phytochemical and palynological data support Acacia as a
polyphyletic taxon and correspond to the molecular evidence10. Furthermore the analyses
show Acacia subg. Acacia as monophyletic and distinct from the other two subgenera, which
is also supported by a study of seed protein profiles11.
In the New World, Acacia subg. Acacia is represented by 57 taxa, which are
characterized by their stipular thorns, leaves with well-developed extrafloral nectaries, pollen
arranged in 16-grain polyads, and a single pollen colporate with columellae. This subgenus
turned out difficult to subdivide on the basis of morphological characters19,20. The same
remains true even for molecular analyses: A phylogeny based on trnK/matK sequences1,16,18
shows a week resolution, although the subg. Acacia per se is well supported and all New
World species form a monophyletic group.
All species of Acacia included in our study are neotropical and assigned to subg.
Acacia. In detail, A. farnesiana, A. macracantha, A. pennatula, and A. cochliacantha belong
to the non-myrmecophytic group of this subgenus, while A. chiapensis, A. cornigera, A.
collinsii, A. globulifera and A. hindsii are myrmecophytes. Among these species, only A.
farnesiana, A. pennatula and A. cochliacantha had already been included in molecular
phylogenetic investigations, while none of the obligate myrmecophytes has so far been used
for such studies. We combined trnK intron data presented by Miller and Bayer18 with data
achieved in our own analysis and found that all Acacia species investigated are, as expected,
members of the monophyletic subg. Acacia and are located within the neotropical group
(Supplementary Figure 1). The myrmecophytic Acacia species (constitutive EFN flow) are
the sister group to a monophyly comprising the non-myrmecophytes A. macracantha, A.
pennatula and A. cochliacantha (bootstrap value = 62). A. farnesiana is part of the neighbour
group of these species, together with A. schaffneri and A. caven (bootstrap value = 54).
Although the bootstrap support for these relationships is low, a paraphyly of the induced EFN
flow with regard to constitutive EFN flow is indicated.
The sister group to Acacia subg. Acacia is formed by Prosopis julifora and Leucaena
leucocephala (inducible EFN flow, both Mimoseae), while Piptadenia flava (also inducible)
is forming an own clade. The monophyly of Acacia subg. Acacia has a high bootstrap support
(100), it is thus highly unlikely that the Mimoseae investigated can be considered as a part of
this monophyly.
The genus Mimosa, which is used in most studies as outgroup for Acacia, forms part
of tribe Mimoseae and is therefore of limited value as an outgroup when phylogenetic
relationships among the different groups of this tribe and the various subgenera of Acacia are
considered17. The placement of the taxa investigated in our study in the phylogeny presented
by Luckow et al.17 is shown in Supplementary Figure 2. In this analysis, which has used a
member of the Caesalpinioideae rather than of the Mimosoideae as the outroup, Piptadenia
flava appears more closely related to Mimosa and Acacia subg. Acacia than Prosopis juliflora
and Leucaena leucocephala. According to this analysis, inducible EFN flow occurs both in
different lineages within the Mimoseae and in Acacia subg. Acacia. Inducible EFN flow via
the same biochemical pathway as reported here has recently been reported for Phaseolus
lunatus21, a member of subfamiliy Faboideae rather than Mimosoideae (Supplementary Figure
2). The inducible EFN secretion in Phaseolus therefore appears homologous to EFN induction
in the Mimosoideae, indicating that this pattern has a wide distribution in the whole family of
Fabaceae.
As to our knowledge, the species investigated in this study are the only mimosoid
species for which the inducibility of EFN flow has been studied. Species with constitutive
EFN flow are closely related to species with inducible EFN flow, and the latter trait also
characterizes members of other phylogenetic groups. This supports the hypothesis that the
constitutive EFN flow has evolved from the inducible state.
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