Biochemical Systematics and Ecology 28 (2000) 53}60
Seed components and taxonomy
of some Acacia species
Alicia L. Lamarque!,*, ReneH e H. Fortunato",
DamiaH n M. Maestri!, Carlos A. GuzmaH n!
!Ca& tedra de Qun& mica Orga& nica, Facultad de Ciencias Exactas, Fn& sicas y Naturales, IMBIV, CONICET,
Universidad Nacional de Co& rdoba, Av. Ve& lez Sarsxeld 1600, 5000 Co& rdoba, Argentina
"CONICET, at Instituto de Recursos Biolo& gicos, INTA, 1712 Castelar, Buenos Aires, Argentina
Received 1 December 1998; accepted 1 March 1999
Abstract
Ten Acacia species belonging to the subgenera Acacia and Aculeiferum were examined for
their seed proximate compositions and fatty acid pro"les. The fatty acids were determined by
GC and GC}MS. Some of the taxa analysed were found to contain hexacosanoic acid as well as
other common fatty acids. A numerical analysis suggested a partial separation among the taxa
belonging to subgenera Acacia and Aculeiferum. Acacia emilioana appears related with species
of subgenus Aculeiferum. ( 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Acacia; Mimosoideae; Fabaceae; Seeds; Fatty acids; Cerotic acid; Chemotaxonomy
1. Introduction
The contribution of morphological, anatomical, ontogenetic and some phytochemical characters has been useful for the classi"cation of the genus Acacia (Bentham,
1842,1862,1875; Vassal, 1972,1981; Evans et al., 1977,1993; Guinet and Vassal, 1978;
Pedley, 1986; Guinet, 1990). In spite of this, there is still some controversy regarding
the infrageneric taxonomy.
The fatty acid composition of seeds has been frequently used as a tool in biochemical systematics and it has proved to be valuable in studies of some plant groups
(Graham et al., 1981; Graham and Kleiman, 1987; Carreras et al., 1989; Ra"i et al.,
* Corresponding author. Tel.: #54-51-334-141; fax: #54-51-334-439.
0305-1978/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 0 3 7 - X
54
A.L. Lamarque et al. / Biochemical Systematics and Ecology 28 (2000) 53}60
1991; Maestri and GuzmaH n, 1995). Acacia species related by their morphology give
oils which are not completely related by their chemistry. Seed oils of some Acacia
species from Australia, India and Africa are known to contain epoxy acids (Earle and
Jones, 1962; Gunstone et al., 1968; Chowdhury et al., 1983; Mannan et al., 1983;
Farooqui et al., 1987; Jamal et al., 1987; Banerji et al., 1988; Prasad and Azeemoddin,
1996), while in the American species studied these fatty acids are not present (Earle
and Jones, 1962; Barklay and Earle, 1974). Brown et al. (1987) have suggested that the
Australian species of this genus may be taxonomically distinct from Acacia species
originating elsewhere in the world according to their fatty acid composition.
In the present investigation we examined the extent to which some seed chemical
components may be useful for taxonomic purposes in Acacia. Five of the species
investigated belong to subgenus Acacia (series Gummiferae of Bentham), four to
subgenus Aculeiferum (series Vulgare of Bentham) and A. emilioana, a taxon recently
described (Fortunato and Cialdella, 1996), does not fully "t in the infrageneric
position proposed for the genus.
2. Materials and methods
Plant Material: Acacia seeds were collected at full maturity in di!erent environmental regions of Argentina, Chile and Paraguay. Details of the plant materials used
are given in Table 1. Voucher specimens of each species from each locality were
deposited in the Herbarium of the Museo BotaH nico (CORD) of the Universidad
Nacional de CoH rdoba, and in the Herbarium of the Instituto de Recursos BioloH gicos,
INTA, Castelar (BAB).
Seed Analysis: Moisture, fat, protein (N]6.25), ash content were determined according to the American Oil Chemists' Society (AOCS) methods (1980).
Fatty Acid Analysis: The fatty acid methyl esters of total lipids were obtained by
direct transmethylation (Carreras et al., 1989). GC analysis was performed on
a SUPELCO-WAX fused silica capillary column (25 m]0.25 mm), from 180 to 2403C
at 53C min~1. Injector and FID detector temp. 2503C; split ratio 1 : 1000. Fatty acid
methyl esters were identi"ed by cochromatography with authentic methyl esters
standards and by GC}MS using a DB5 (SE54) capillary column (15 m]0.25 mm)
from 80 to 2903C (injector and FID detector temp. 2903C; split ratio 1 : 1000). The
interface temp. was 2903C and the ionization voltage 70 eV. The fatty acid composition was expressed as percentage of total fatty acids. Spectra were recorded in
combination with the data system NIST.
IR analysis: The oils and fatty acid methyl esters were qualitatively examined for the
presence of epoxy fatty acids by IR. The IR spectra were obtained in KBr discs.
Duplicate analysis of chemical attributes were performed for each sample.
Numerical Analysis: A cluster analysis was performed with seed components and
fatty acid composition using the (Numerical Taxonomy System of Multivariate
Statistical Programs (NTSYS-pc) (Rohlf, 1987). A dendrogram was constructed by the
Unweighted Paired Group Method of Arithmetic Averages (UPGMA) method of
Sneath and Sokal (1973).
A.L. Lamarque et al. / Biochemical Systematics and Ecology 28 (2000) 53}60
55
Table 1
Collection data of Acacia species
Species
Location
Subgenus Acacia
Acacia aroma Gill. ex Hook (1)
Acacia aroma (2)
Acacia aroma (3)
Acacia aroma (4)
Acacia aroma (5)
Acacia atramentaria Benth.(1)
Acacia atramentaria (2)
Acacia atramentaria (3)
Acacia caven (Mol.) Mol. var caven (1)
Acacia caven (2)
Acacia caven (3)
Acacia caven (4)
Acacia horrida (L.) Willd.
Acacia macracantha Humf. et Bompl. ex Willd.
CoH rdoba: Route 28 Km 873 (CORD)
San Juan: Route 12 Km 87 (CORD)
San Juan: Route 12 Km 66 (CORD)
CoH rdoba: Villa Allende (BAB)
CoH rdoba: Villa Allende (BAB)
CoH rdoba: Capital (BAB)
CoH rdoba: Route 38 (BAB)
San Juan: Route 12 Km 90 (CORD)
CoH rdoba: La Calera (CORD)
CoH rdoba: Carlos Paz (BAB)
Salta: Cachi (CORD)
CoH rdoba: Villa Allende (BAB)
Chile: Route 5 Km 80 (BAB)
Salta: Capital (BAB)
Subgenus Aculeiferum
Acacia emilioana Fortunato & Ciald.
Acacia furcatispina Burk. (1)
Acacia furcatispina (2)
Acacia furcatispina (3)
Acacia parviceps (Speg.) Burk
Acacia praecox Griseb. (1)
Acacia praecox (2)
Acacia visco Lor. ex Griseb
Alto Paraguay: Cerro Cabrera (BAB)
San Juan: Route 12 Km 87 (CORD)
CoH rdoba:El Diquecito (BAB)
CoH rdoba: Capital (BAB)
Salta: Capital (BAB)
CoH rdoba:El Diquecito (CORD)
Santiago del Estero: 2 Km W from Villa La Punita
(BAB)
Salta: Valles CalchaqumH es (CORD)
3. Results and discussion
Seed chemical components of Acacia oils are given in Table 2. The protein content
varied between 22.7 and 37.1%, A. visco had the highest value. The oil content
constituted more than 4.9% in all species, reaching a value of 21.6% in A. parviceps.
The ash content ranged from 3.3 to 4.5%. The seed oil characteristics compare well
with those of other Acacia species (Brown et al., 1987; Jamal et al., 1987; Banerji et al.,
1988).
3.1. IR spectra analysis
The IR spectra of the oil and their respective mixed methyl esters did not show
bands at 840 and 820 cm~1 attributable to the epoxy group (Daulatabad and
Hosamani, 1991).
56
A.L. Lamarque et al. / Biochemical Systematics and Ecology 28 (2000) 53}60
Table 2
Seed proximate composition from Acacia species
Species
Moisture (%)
Fat (%)
Protein (%)
Ash (%)
Subgenus Acacia
Acacia aroma (1)
Acacia aroma (2)
Acacia aroma (3)
Acacia aroma (4)
Acacia aroma (5)
Acacia atramentaria (1)
Acacia atramentaria (2)
Acacia atramentaria (3)
Acacia caven (1)
Acacia caven (2)
Acacia caven (3)
Acacia caven (4)
Acacia horrida
Acacia macracantha
7.2$0.3
7.8$0.3
8.7$0.2
9.1$0.5
9.4$0.2
9.3$0.5
10.0$0.7
9.9$0.7
4.4$0.5
4.3$0.2
4.6$0.2
4.6$0.2
8.4$0.2
9.8$0.6
8.3$0.8
6.9$0.8
6.8$0.7
7.8$0.6
8.1$0.6
7.4$0.2
4.9$0.2
6.7$0.3
8.1$0.2
8.2$0.4
8.2$0.3
8.0$0.3
12.5$0.6
8.6$0.4
28.5$0.8
27.9$0.9
28.1$0.7
27.5$0.9
26.9$0.8
24.1$0.5
23.2$0.2
23.7$0.1
26.8$0.6
22.7$0.3
23.9$0.8
25.3$0.5
25.5$1.2
23.1$0.2
3.9$0.1
3.8$0.1
3.6$0.2
4.3$0.2
3.3$0.1
3.8$0.1
3.7$0.1
3.7$0.2
3.8$0.1
3.7$0.2
3.8$0.2
4.0$0.3
4.5$0.1
3.3$0.2
Subgenus Aculeiferum
Acacia emilioana
Acacia furcatispina (1)
Acacia furcatispina (2)
Acacia furcatispina (3)
Acacia parviceps
Acacia praecox (1)
Acacia praecox (2)
Acacia visco
7.8$0.5
7.9$0.3
7.4$0.5
8.0$0.4
10.0$0.3
6.7$0.1
6.3$0.5
4.2$0.1
9.0$0.5
13.0$0.9
11.9$1.05
18.9$0.4
21.6$0.6
8.3$0.3
10.5$0.2
7.7$0.5
26.9$0.9
25.1$0.7
26.2$0.7
27.6$0.9
23.9$0.8
27.3$0.3
26.6$0.5
37.1$0.3
3.3$0.1
4.0$0.1
3.8$0.2
3.3$0.3
4.3$0.4
4.2$0.1
3.9$0.1
3.9$0.3
Mean$standard deviation.
3.2. GC-MS
The fatty acid methyl esters of the oils are presented in Table 3. Thirteen fatty acids
were identi"ed. The unsaturated acids included 9-octadecenoic (18:1), 9,12-octadecadienoic (18:2), and 9,12,15-octadecatrienoic (18:3) acids as the principal components. Besides these constituents, 9-hexadecenoic (16:1), 9-eicosenoic (20:1) and
9-docosenoic (22:1) acids were detected in small quantities.
The lowest percentage of 18:1 acid (2.7%) was detected in A. emilioana, while in the
other species this fatty acid comprised 12.9}31.5% of the total fatty acids. The 18:3
acid percentage reached about 21% in Acacia praecox, whereas the 18:2 values were in
the range 35.3}60.3%.
Saturated acids were mainly composed of hexadecanoic (16 : 0) and octadecanoic
(18 : 0) acids,while hexacosanoic fatty acid (26 : 0) was absent in Acacia horrida and
A. emilioana. Acacia emilioana presented an exceptionally high percentage of 16:0
acid (42.8%) resembling in this aspect some species of the Old World, like A. torta
(36.7%), A. planifrons (31.3%), A. senegal (36.4%) and A. crassicarpa (35%) (Brown
et al., 1987; Jamal et al., 1987; Banerji et al., 1988). Fortunato and Cialdella (1996) have
A.L. Lamarque et al. / Biochemical Systematics and Ecology 28 (2000) 53}60
57
Table 3
Fatty acid composition (% of total fatty acids) of seed oils from Acacia species
Species
14 : 0 16 : 0 18 : 0 20 : 0 22 : 0 24 : 0 26 : 0 16 : 1 18 : 1 18 : 2 18 : 3 20 : 1 22 : 1
Subgenus Acacia
Acacia aroma (1)
tr
Acacia aroma (2)
tr
Acacia aroma (3)
tr
Acacia aroma (4)
tr
Acacia aroma (5)
0.2
Acacia atramentaria (1) 0.1
Acacia atramentaria (2) 0.1
Acacia atramentaria (3) 0.1
Acacia caven (1)
0.2
Acacia caven (2)
tr
Acacia caven (3)
0.1
Acacia caven (4)
tr
Acacia horrida
tr
Acacia macracantha
2.3
11.9
11.7
11.6
11.5
12.0
9.3
8.1
7.9
12.7
11.0
11.5
12.1
11.3
13.0
6.2
5.8
5.3
4.6
5.3
3.2
3.8
1.4
5.9
4.4
4.7
5.2
1.8
4.6
1.9
2.0
1.5
2.2
1.9
1.1
1.0
1.1
1.1
1.1
1.2
1.1
0.8
2.9
1.3
1.4
1.4
1.5
1.3
0.8
0.9
0.9
0.6
0.8
0.7
0.7
1.3
1.0
0.2
0.2
0.2
0.3
0.2
0.4
0.3
0.8
0.3
tr
0.2
tr
0.5
0.3
0.6
0.5
0.5
0.6
0.5
0.2
0.1
0.6
2.1
1.6
1.8
1.7
}"
tr!
0.3
0.2
0.2
0.1
0.3
0.2
0.3
0.4
0.4
tr
0.3
0.2
tr
1.0
26.0
23.9
24.4
20.7
24.7
28.5
24.3
27.0
31.5
29.1
30.2
30.3
21.1
25.8
50.6
53.3
53.6
56.0
52.1
55.6
60.3
58.4
44.4
51.6
48.9
48.3
59.1
46.0
0.4
0.5
0.8
1.7
0.6
tr
0.4
0.5
0.2
tr
0.1
0.1
3.0
0.8
0.4
0.3
0.3
0.5
0.4
0.3
0.3
0.4
0.1
0.1
0.1
0.1
0.8
1.4
tr
tr
tr
tr
tr
tr
tr
0.2
tr
tr
tr
tr
}
tr
Subgenus Aculeiferum
Acacia emilioana
Acacia furcatispina (1)
Acacia furcatispina (2)
Acacia furcatispina (3)
Acacia parviceps
Acacia praecox (1)
Acacia praecox (2)
Acacia visco
42.8
23.6
22.9
23.3
16.3
5.7
5.6
10.0
5.2
7.8
8.3
6.8
5.5
2.2
2.1
3.0
1.5
1.1
1.1
1.0
12.2
0.2
0.3
1.7
1.0
0.4
0.3
0.4
3.9
0.4
0.7
2.6
0.3
0.6
0.5
0.6
2.6
0.3
0.2
0.5
}
tr
tr
tr
tr
tr
tr
0.2
0.5
0.8
0.8
0.6
2.1
tr
tr
0.4
2.7
18.6
19.1
20.0
17.9
13.3
12.9
25.6
44.2
44.7
44.8
45.0
35.3
56.3
57.1
53.2
0.2
0.9
0.8
0.9
tr
21.3
20.9
2.2
1.4
0.5
0.4
0.4
tr
tr
tr
0.5
}
0.3
0.3
0.3
}
tr
tr
}
0.2
0.4
0.3
0.4
4.1
tr
tr
tr
!tr, trace (0.1%.
"}, not detected.
demonstrated that, from an exomorphological point of view, A. emilioana is related to
other Acacia species from Africa and Asia. In spite of these interesting similarities
between A. emilioana and the Old World species, the former lacks epoxy fatty acids
unlike the species cited above.
Fatty acid analyses of seeds from approximately 50 Acacia species have been
reported (Earle and Jones, 1962; Gunstone et al., 1968; Barklay and Earle, 1974;
Chowdhury et al., 1983; Mannan et al., 1983; Rivett et al., 1983; Brown et al.,
1987; Farooqui et al., 1987; Jamal et al., 1987; Banerji et al., 1988; Prasad and
Azeemoddin, 1996). By comparison, the seed oils of all American species investigated,
including the present study, lacked epoxy fatty acids. Consequently, the sporadic
occurrence of epoxy fatty acids in seed oils of non-American Acacia species,
grouped in di!erent subgenera according to Vassal's classi"cation, substantiates
the suggestion that the epoxy fatty acids could be interpreted as new `chemical
markersa in these taxa. This, however, has still to be augmented by the analysis
58
A.L. Lamarque et al. / Biochemical Systematics and Ecology 28 (2000) 53}60
Fig. 1. Phenogram showing relationships of 21 samples of Acacia prepared on the basis of similarity
coe$cient 17 characters.
of additional species, in order to support a reexamination of the relationships within
the genus.
3.3. Numerical analysis
The signi"cance of the taxonomic value of chemical data for the classi"cation of
Acacia species can be seen in Fig. 1. The phenogram constructed with 17 characters
from seed composition and fatty acid percentages suggests a partial separation of the
subgenera. It shows the presence of one major group (consisting of A. caven, A. aroma,
A. atramentaria, A. horrida and A. macracantha: Subg. Acacia), and the rest of the
species (Subg. Aculeiferum) are linked at di!erent taxonomic distances. In the main
group, A. macracantha is segregated and no close relationship with A. aroma could be
seen; therefore, these species must remain as two di!erent taxa until further investigation can be done as recommended by Cialdella (1984). Within the Subg. Aculeiferum,
A. parviceps is the most peripherical species. The occurrence of high percentages of oil
and 20 : 0 acid and relatively low levels of 18 : 1 and 18 : 2 acids, makes a di!erence
between this species and the others. On the other hand, Acacia emilioana appears to be
more closely related to A. praecox than to A. furcatispina in agreement with the
observations of Fortunato and Cialdella (1996).
Acknowledgements
Financial support for this work was provided by CONICET and CONICOR.
A.L. Lamarque et al. / Biochemical Systematics and Ecology 28 (2000) 53}60
59
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Damián Maestri