Biochemical Systematics and Ecology 38 (2010) 538–547
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Biochemical Systematics and Ecology
journal homepage: www.elsevier.com/locate/biochemsyseco
Essential oil composition of nine Apiaceae species from western United
States that attract the female Indra Swallowtail butterfly (Papilio indra)
Vasu Dev a, Wayne H. Whaley b, *, Sarah R. Bailey a, Eric Chea a, Jeannie G. Dimaano a,
Dhara K. Jogani a, Bill Ly a, Dennis Eggett c
a
Department of Chemistry, California State Polytechnic University, Pomona, CA 91768, USA
Department of Biology, College of Science and Health, Utah Valley University, 800 W. University Parkway, Orem, UT 84058, USA
c
Department of Statistics, Brigham Young University, Provo, UT 84662, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 September 2009
Accepted 29 May 2010
Aletes acaulis, Cymopterus hendersonii, Cymopterus panamintensis var. acutifolius, Lomatium
rigidum, Lomatium scabrum var. tripinnatum, Musineon tenuifolium, Sphenosciadium capitellatum, Tauschia arguta and Tauschia parishii are among the twenty-two species of the
Apiaceae family to which female Indra Swallowtail butterflies (Papilio indra: Lepidoptera)
are attracted for oviposition. Because plant volatile oils are known to be attractants for
female butterflies, the percent composition of the essential oils of each species was
studied. Amongst the nine host plants 168 essential oil components were identified representing between 84% and 99% of the oils. Principal Components Analysis and hierarchical
cluster analysis on the essential oil compositions of the larval host plants against four nonlarval host plants separated the hosts from the non-hosts into distinct clusters. Volatile
components of the oils common to the nine species of Apiaceae are correlated with the
expression of physiological attraction behavior by the butterfly.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Apiaceae
Essential oil composition
Indra Swallowtail butterfly
Papilio indra
Aletes
Cymopterus
Lomatium
Musineon
Sphenosciadium
Tauschia
1. Introduction
Butterflies in the genus Papilio (Lepidoptera: Papilionidae) are known to be attracted to an appropriate larval host plant by
olfactory cues emitted from certain volatile compounds (Baur et al., 1993; Feeny et al., 1989; Heinz, 2008). Honda’s (1995)
review of oviposition behavior across the spectrum of the Lepidoptera indicated that females generally utilize plant volatiles for orientation to appropriate host plants. The Indra Swallowtail butterfly (Papilio indra) has been observed to oviposit on
twenty-two species in the family Apiaceae (>275 species in western U.S.; Cronquist et al., 1997; Hickman, 1993; Hitchcock and
Cronquist, 1973; Kearney et al., 1960). The butterfly is found in widely scattered populations in mountainous regions across
the western U.S. and generally one, rarely two, of these host plants are utilized by any one population (Whaley, 2000). At
several locations in the Great Basin and Mojave Desert mountain ranges its larval host plants grow as widely scattered singles
tucked within rock crevices to conserve moisture, which often makes them difficult for researchers to find (e.g., Cymopterus
panamintensis). However, the female’s sensitive antennae sensilla allow her to find host plants with ease. The butterfly’s
attraction to its host plants has been ascribed to the citrusy/piney aroma of these plants, with just three emitting anise-like or
celery-like aromas (Dev et al., 2007; Whaley, 2000). It is conceivable that other aromatic as well as non-aromatic components
might also be responsible for the butterfly’s behavior. However, whatever attracts the butterfly to the plant in the first place, it
* Corresponding author. Tel.: þ1 801 863 8607; fax: þ1 801 863 8064.
E-mail address: wwhaley@uvu.edu (W.H. Whaley).
0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2010.05.010
V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
539
drums around the plant with its foretarsi prior to ovipositing its eggs. The drumming behavior of the butterfly prior to
ovipositing has been observed to involve compounds other than essential oil components (Feeny et al., 1988). Our assumption
of the involvement of certain components of the essential oils of the plants in attracting the butterfly has lead us to the study
of the composition of these oils in its Apiaceae larval host plants (Beauchamp et al., 2009; Dev et al., 2007) anticipating some
consistencies in oil components across the hosts. In the present communication we report the essential oil compositions of
nine more host species of Apiaceae and infer which compounds in these oils might be involved in P. indra female attraction.
2. Materials and methods
2.1. Plant material
Living plant specimens were collected at the locations indicated below and kept cold under ice during their transportation
to the laboratory. They were deposited at the indicated herbaria and their accession numbers assigned. Specimens were
collected in the flowering or seed stages, either of which is known to be used for oviposition by Indra Swallowtail butterfly.
Aletes acaulis was collected along Clear Creek Cyn, (north side of Mt. Zion, Lookout Mountain), Jefferson Co., Colorado, T4S,
R71 W Sec. 2, elev. 7000 ft. (access. no. COLO 451097).
Cymopterus hendersonii was collected from two locations to determine any differences in the compositions of their
essential oils.
Chen-1: Carbon Co., Montana, Beartooth Plateau, at Rock Creek Vista along Hwy. 212; 45 020 3300 N, 109 240 2400 W.
elev.9170 ft. (access no. UM 78489). Chen-2: Utah Co., Utah, summit of the glacial cirque at head of Mineral Basin, T3S, R3E sec.
18 (SE ¼), elev.10 200 ft. (access. no. BYU 234170).
Cymopterus panamintensis var. acutifolius was collected off Rabbit Spring Road near where Highway 18 ends and Highway
0
247 starts, Granite Mountains, San Bernardono Co., California; GPS coordinates lat. 34 28 N, long. 116 590 W, elev. 3219 ft.
(access. no. UVSC 13100).
Lomatium rigidum was collected in Bishop Canyon along Highway168 at1.3 miles below South Lake Road junction, Inyo Co.,
California, 37160 3000 N, 118 340 3200 W, elev. 7415 ft. (access. no. RSA 745006).
Lomatium scabrum var. tripinnatum was collected along highway18 at 1.3 miles north of St. George, Washington Co., Utah,
37 80 44.8600 N, 113 360 4.0600 W., elev. 3207 ft. (access. no. UVSC 13112).
Musineon tenuifolium was collected along Highway 44, ca. 8 miles west of Rapid City, Pennington Co., South Dakota. 44
0
03 31.5200 N, 103 21051.3900 W, elev. 4197 ft. (access. no. UVSC 13106).
Sphenosciadium capitellatum was collected along Saddleback Lake off Saddleback road turnoff 2.2 miles east of Tioga Pass
entrance to Yosemite National Park from HWY 120, Mono Co., California, 37 580 0300 N, 119 160 2700 W, elev.10 100 ft. (access.
no. UVSC 09936).
Tauschia arguta was collected 3.3 miles west of Mountain Center along San Jacinto South Fork Trail, Riverside Co., California, 33 410 4400 N, 116 450 3900 W, 4000 ft elev. (access. no. UVSC 13102).
0
00
Tauschia parishii was collected at Butterbredt Peak, Jawbone Canyon Road, Kern Co., California, GPS coordinates 35 23 04
0
00
N, 118 09 22 W, elev. 5800 ft. (access. no. RSA 745005).
2.2. Isolation of oils
The fresh plant material, which included stems, leaves, inflorescence material and fruits, was hydrodistilled and the
respective oils isolated from the aqueous distillates by procedure described in an earlier communication (Dev et al., 2007).
2.3. Qualitative and quantitative analyses
A Hewlett Packard 6890 GC fitted with a FID and a HP5MS capillary column (30 m 0.25 mm, film thickness 0.25 mm) was
used for determining composition and Kováts retention indices (RI). The oven temperature was programmed at 50 C for
10 min and then 3 C/min to 240 C, after which it was maintained at this temperature for 5 min. The GC/MS analyses were
conducted with an AGILENT 5973 Network Mass Selective Detector interfaced with AGILENT 6950 GC system fitted with
a capillary column matching the one used with the HP 6890 GC. The GC of the GC/MS was programmed at 50 C for 10 min
followed by 3 C/min to 230 C and then isothermal at 230 C for10 min.
Principal Components Analysis (PCA; SAS statistical software Version 9.1) was used to reveal any differences between the
essential oil compositions of the nine P. indra host plants presented here and the previously published essential oil
compositions of four non-host plants, Lomatium dissectum (Bairamian et al., 2004), L. dasycarpum (Asuming et al., 2005), L.
utriculatum (Asuming et al., 2005), and L. nevadense (Beauchamp et al., 2007). These four non-hosts were chosen because they
are prevalent within the butterfly’s habitat, and because genus Lomatium is more commonly used by the butterfly (14 of the
22 host species). The first two PCA loadings were used to determine the important essential oil compounds separating host
from non-host. Then a hierarchical cluster analysis was performed on the first six PCA loadings to group the plants into
potential phylogeny.
540
V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
3. Results
3.1. Volatile components of the essential oils and host plant aromas
The data with respect to the yield of oil from each plant specimen are listed in Table 1. The percent composition of the oil
components are reported in Table 2 as computer read-out without correction factors and rounded to the first decimal place.
Identified compounds accounting for less than 0.1% are labeled as trace (tr) components. The identification of the oil
components was established by an excellent match of both their RI as well as mass spectra with the RI and mass spectra of the
corresponding compounds available in the literature as listed under Identification Methods. Among the nine host species 168
compounds were identified. The percentage of the identified components varied from >84% for M. tenuifolium to >95% for C.
hendersonii. Therefore, it is possible that some compounds important for attraction may have been missed.
With the exception of C. hendersonii, each of the larval host plants had a citrus like or lemon/orange aroma and taste. The C.
hendersonii had a characteristic anise aroma and taste. For the nine species the majority of the citrusy aroma was due to the bphellandrene þ limonene and the pinene (Table 1). For the two samples of C. hendersonii, methyl chavicol and (E)-anethole
produce its anise aroma. When evaluating each plant’s aroma it was sometimes difficult to distinguish a citrusy aroma from
a piney aroma.
3.2. Principal Components Analysis and cluster analysis
The receptor neurons of some lepidopterans are tuned narrowly and with a high sensitivity to a particular plant volatile
(Hansson, 1995, 2002). Because the butterfly’s olfactory threshold is suspected to be narrowly tuned to the same or similar
compounds, only the most prevalent essential oil components were used for the statistical analysis. These were determined to
be those components of the oils that were present in either at least 5 out of the 10 host plants or at least 2 of the four non-host
plants. Eighty three of the168 compounds in the host plant and non- host plant essential oils met this criterion. We found
through preliminary principal component analysis that if there was a large discrepancy in concentration levels of an essential
oil compound used in our analysis, for example 30%–0.2% for myrcene, that those plants showing small amounts were treated
as if they were effectively the same as zero. This did not seem to be the correct way of handling the large discrepancies in
concentrations and did not support the objectives. Therefore, we felt that using presence or absence would be more
representative of the makeup of each plant. Thus, PCA on the presence or absence of these 83 compounds was used to
distinguish the nine larval host plants from the four non-host plants.
The PCA results showed that the first six principal components explained 77% of the variance in the data. The cluster tree
produced by using these principal components revealed that the essential oil compositions of the four non-host species
differed significantly (distance >1.00) from the host species (Fig. 1). The C. hendersonii samples from Montana and Utah
populations were most similar in essential oil constituents. However, the Lomatium species and the two Tauschia species
differed substantially in essential oil composition (Fig. 1).
4. Discussion
4.1. Non-polar and polar essential oil components as stimulants
Most species of butterflies are oligophagous, but their egg laying habits are usually restricted to species within one or a few
related plant families. A majority of the 200 þ species of Papilio use plants in the Rutaceae (citrus family), the family
considered ancestral to the genus (Scriber, 1973), but eight species comprising what is referred to as the Papilio machaon
species group, which includes P. indra, use members of the Apiaceae (the umbels) as larval hosts. In the papilionids attraction
to an appropriate host is by olfactory cues from volatile compounds (Baur et al., 1993; Feeny et al., 1989; Saxena and Goyal,
1978), and to some degree by leaf shape recognition (Papaj, 1986; Rausher, 1978), but to date only one member of the machaon
Table 1
Yields of oils from Aletes acaulis (Aaca),Cymopterus hendersonii (Chen-1 and Chen-2),Cymopterus panamintensis (Cpan), Lomatium rigidum (Lrig), Lomatium scabrum var. tripinnatum (Lsca), Musineon tenuifolium (Mten), Sphenosciadium capitellatum (Scap),Tauschia arguta (Targ), and Tauschia parishii (Tpar).
Plant name
g plant
mg oil
% oil
Aaca
Chen-1
Chen-2
Cpan
Lrig
Lsca
Mten
Scap
Targ
Tpar
104
77
65
50
73
273
225
173
180
480
129
79
1066
254
68
430
63
156
489
756
0.12
0.10
1.64
0.51
0.09
0.16
0.03
0.09
0.27
0.16
541
V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
Table 2
Composition of the essential oils of Aletes acaulis (Aaca), Cymopterus hendersonii (Chen-1, and Chen-2), Cymopterus panamintensis (Cpan), Lomatium rigidum (Lrig),
Lomatium scabrum (Lsca), Musineon tenuifolium (Mten), Sphenosciadium capitellatum (Scap), Tauschia arguta (Targ), and Tauschia parishii (Tpar).
Essential Oil Components
RI(obs)
Aaca
Chen-1
Chen-2
Cpan
Lrig
Lsca
Mten
Scap
Targ
Tpar
ID Methoda
ethyl 2-methylbutyrate
(E)-2-hexenal
isobutyl isobutyrate
a–thujene
a–pinene
camphene
benzaldehyde
sabinene
b-pinene
myrcene
a–phellandrene
isobutyl 2-methylbutyrate
isobutyl 3-methylbutyrate
d-3-carene
(E)-2-hexenyl acetate
isobutyl isovalerrate
a–terpenene
2-methylbutyl isobutyrate
p-cymene
b-phellandrene þ limonene
1,8-cineole
(Z)-b-ocimene
butyl 2-methylbutyrate
(E)-b–ocimene
isobutyl angelate
g–terpinene
cis-sabinene hydrate
m-cresol
cis-linalool oxide (furanoid)
butyl angelate
terpinolene
6,7-epoxymyrcene
6-camphenone
linalool
3-methylbutyl 2- methylbutanoate
2-methylbutyl 2- methylbutanoate
3-methylbutyl 3- methylbutanoate
2-methylbutyl 3-methylbutanoate
dehydrosabina ketone
allo-ocimene
camphor
3-methyl-2-buten-1-yl 2-methylbutyrate
hexyl isobutyrate
isoamyl angelate
citronellal
2-methylbutyl angelate
borneol
lavandulol
4-terpineol
viridine
naphthalene
p-cymenene-8-ol
cryptone
amyl angelate
a-terpineol
(E)-4-decenal
(Z)-undecenal
methyl chavicol
2-methylbutyl tiglate
4-methylpentyl 2-methylbutyrate
g–terpineol
decanal
citronellol
thymol methyl ether
neral
cuminal
carvacrol methyl ether þ hexyl 3-methylbutyrate
carvone
piperitone
854
856
921
932
938
953
963
978
979
993
1002
1005
1009
1010
1011
1014
1017
1021
1027
1031
1034
1043
1048
1054
1058
1062
1070
1076
1076
1083
1088
1094
1096
1100
1102
1106
1107
1112
1122
1132
1146
1146
1153
1154
1155
1158
1167
1171
1176
1178
1181
1186
1188
1190
1192
1194
1195
1196
1200
1201
1202
1207
1228
1236
1241
1242
1247
1247
1254
–
–
–
0.2
10.8
0.2
–
–
38.3
8.1
0.1
–
–
–
–
–
0.1
–
0.2
29.0
0.1
1.5
–
0.5
–
0.3
–
–
–
–
0.3
–
–
0.5
tr
–
–
0.1
–
tr
–
–
tr
–
–
–
tr
–
0.5
–
–
–
0.1
–
1.1
–
–
0.2
–
–
–
–
0.3
tr
–
–
–
–
–
–
–
0.8
0.2
0.3
0.1
–
11.3
0.7
2.1
tr
0.2
0.6
–
–
–
0.6
0.2
0.6
2.7
0.1
0.2
tr
3.1
–
1.8
0.1
–
–
–
0.2
–
–
1.2
–
–
0.3
–
0.1
–
–
–
0.3
–
1.2
–
–
–
0.8
–
–
–
–
–
0.7
–
–
29.9
–
–
–
–
2.7
0.4
tr
–
0.4
–
0.3
tr
–
tr
0.1
0.6
tr
0.04
7.8
1.2
1.6
tr
tr
0.1
–
–
–
0.2
–
tr
4.7
0.1
0.2
tr
2.9
–
0.5
–
–
–
–
tr
–
–
–
–
tr
0.1
0.1
0.1
–
–
–
–
–
0.9
–
–
–
0.6
–
–
–
–
–
0.6
–
–
42.3
–
–
0.7
–
3.5
0.1
0.2
–
tr
–
0.3
0.8
0.8
–
0.1
0.9
0.1
–
0.6
0.9
30.0
0.2
–
–
–
–
–
tr
–
0.2
19.8
–
4.9
–
4.5
–
tr
–
–
0.1
–
0.2
0.1
–
3.6
–
–
–
–
0.1
0.1
0.3
–
–
–
0.9
–
–
0.1
0.3
–
–
–
0.4
–
0.2
–
0.5
0.2
0.3
–
–
0.6
1.6
–
–
–
–
–
–
–
tr
–
0.1
6.9
0.9
–
0.1
3.5
3.9
0.6
0.1
–
–
–
–
–
–
0.9
28.6
–
0.2
–
0.6
0.2
0.1
–
–
–
0.3
0.8
–
0.3
0.8
tr
1.1
–
0.1
0.2
–
0.4
–
–
0.2
0.1
0.9
0.1
–
0.2
–
–
–
5.6
0.3
0.1
–
–
0.8
–
0.4
–
–
0.5
–
–
0.5
–
–
–
–
1.4
0.1
–
–
4.6
8.2
0.7
0.5
–
0.1
–
0.1
0.6
0.1
–
0.4
0.1
1.0
0.1
tr
–
–
–
–
–
–
0.1
25.5
–
0.3
–
0.4
–
0.3
–
0.1
–
tr
0.2
–
–
2.3
0.3
0.1
tr
–
–
–
–
0.2
–
–
0.4
–
–
–
0.4
–
–
–
0.5
–
0.4
0.9
–
–
–
–
–
1.2
2.4
–
–
–
–
–
–
–
tr
–
0.1
1.0
–
–
0.2
tr
0.2
5.0
–
–
0.6
–
0.5
0.1
–
2.7
11.3
–
17.9
–
4.3
–
6.4
–
–
–
–
1.2
–
–
1.2
tr
0.6
2.0
3.8
–
0.3
–
–
–
–
–
–
–
0.3
–
0.4
–
–
–
–
0.5
–
–
0.1
2.4
3.0
1.0
–
–
1.6
–
–
0.3
–
0.4
6.4
–
13.1
–
5.7
–
0.2
–
0.2
–
–
9.2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.2
0.7
–
–
0.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.1
–
0.3
6.4
0.6
–
0.4
8.8
2.6
2.0
–
–
–
–
–
0.1
–
0.1
39.4
–
1.3
–
1.5
–
1.2
–
–
–
–
0.8
–
–
–
0.3
0.6
0.1
0.2
0.1
tr
0.1
–
–
–
–
0.5
–
–
0.1
–
–
–
0.1
0.4
0.1
–
–
0.1
–
–
–
tr
0.5
–
–
0.1
–
–
–
C
A
C
A
A,B
A
A,B
A
A,B
A,B
A
B
B
A
A
A
A
C
A,B
A,B
A,B
A
B
A
B
A
A
A
A
B
A
A
A
A,B
A,B
A.B
B
B
A
A
A,B
B
A
B
A
B
A
A
A
A
A
A
A
B
A
A
A
A
B
B
A
A
A
A
A
A
A
A
A
0.1
0.1
0.6
0.2
–
–
–
0.3
26.0
–
11.8
0.4
4.0
–
0.3
–
–
–
–
0.8
–
–
–
–
1.5
–
–
0.1
0.1
–
–
–
–
0.1
–
–
–
–
–
–
–
0.3
–
0.5
–
–
–
–
–
–
–
0.4
–
–
–
–
–
–
–
–
0.2
0.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
542
V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
Table 2 (continued)
Essential Oil Components
RI(obs)
Aaca
Chen-1
Chen-2
Cpan
Lrig
Lsca
Mten
Scap
Targ
Tpar
ID Methoda
geraneol
linalool acetate
methyl citronellate
geranial
citronelly formate
dihydrolinalool acetate
p-menth-1-en-7-al
a-terminen-7-al
bornyl acetate
C6H11-angelate/tiglate
(E)-anethole
(Z,Z,Z)-3,6,9-tridecatriene
lavandulyl acetate
carvacrol
undecanal
4-methylhexyl 2-methylbutanoate
neoiso-pulegyl acetate
p-vinyl guaiacol
(E,E)-2,4-decadienal
myrtenyl acetate
3-oxo-p-menth-1-en-7-al
trans-carvyl acetate
a-cubebene
eugenol
cyclosativene
longicyclene
a-copaene
daucene
b-bourbonene
b-cubebene
b-elemene
longifolene
methyl eugenol
dodecanal
a-gurjunene
b-ylangene
b–caryophyllene
b-copaene
a-guaiene
2-methylbutyl benzoate
aromadendrene
a–humulene
(E)-b–farnesene
cis-muurola-4(14),5-diene
4,5-diepi-aristolochene
trans-cadina-1(6),4-diene
b-chamigrene
g-muurolene
amorpha-4,7(11)-diene
ar-curcumene
germacrene D
citronellyl isobutyrate
b-selinene
(E)- b-ionone
2-phenylethyl 3-methylbutanoate
a-selinene
valencene þ benzyl tiglate
a–zingiberine
viridiflorene
bicyclogermacrene
a-muurolene
germacrene A
(E,E)-a–farnesene
a-cuparene þ (E,E)-a- farnesene
lavandulyl 2-methylbutyrate
g-cadinene
(Z)-g-bisabolene
d-cadinene
trans-cadina-1(2),4-diene
1258
1258
1264
1274
1275
1277
1277
1283
1288
1289
1290
1291
1294
1300
1306
1307
1314
1314
1322
1334
1336
1339
1348
1360
1370
1372
1376
1380
1388
1388
1393
1406
1407
1409
1412
1419
1422
1432
1438
1442
1448
1453
1459
1468
1474
1477
1477
1479
1481
1484
1484
1484
1488
1489
1493
1496
1496
1496
1497
1498
1500
1502
1509
1510
1512
1516
1516
1526
1534
–
–
–
–
–
–
–
–
–
–
tr
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.1
–
–
–
0.1
–
–
–
tr
–
–
–
–
–
tr
–
–
–
–
1.2
–
–
–
0.3
–
–
–
–
–
–
–
1.0
–
–
0.2
–
–
–
–
0.1
–
–
–
–
–
–
–
–
–
–
–
11.8
–
–
–
–
–
–
tr
–
–
–
–
–
tr
–
–
–
–
–
–
–
–
19.0
–
–
–
0.2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.1
–
–
tr
–
–
–
–
6.1
–
–
0.7
–
–
–
–
–
–
–
–
–
0.3
–
–
–
–
–
–
21.5
–
–
–
–
–
–
tr
0.1
–
–
–
–
0.2
–
–
–
–
–
–
–
–
4.3
–
–
–
0.1
–
–
–
–
tr
tr
–
–
tr
–
–
–
–
–
tr
–
–
–
–
–
0.2
–
–
–
–
0.1
–
–
–
–
–
–
–
1.2
–
–
–
0.1
–
–
0.6
–
–
–
–
–
–
–
–
–
tr
–
0.1
–
–
–
–
–
–
–
0.6
–
0.5
–
–
–
–
–
–
0.1
–
–
–
1.2
–
–
–
tr
–
0.2
–
–
3.5
–
0.2
–
–
–
–
1.2
–
–
0.2
0.4
–
–
–
0.3
–
2.6
–
–
–
0.1
–
–
0.3
–
0.1
2.4
0.2
–
2.0
–
–
–
0.2
–
–
–
–
0.8
–
–
–
0.1
–
0.1
–
0.4
0.5
–
–
–
–
–
0.1
–
3.7
–
–
–
0.6
–
–
–
–
–
–
–
–
–
–
–
–
–
0.1
–
0.5
–
1.0
–
–
–
–
–
1.2
0.1
–
–
–
0.4
–
–
–
–
–
0.3
–
–
2.1
–
–
–
–
–
–
–
–
3.0
0.5
0.3
–
–
–
0.5
–
3.2
0.1
1.5
–
–
–
0.2
–
–
–
1.1
–
–
–
–
–
–
–
–
–
–
–
–
–
1.0
0.3
–
0.1
–
–
–
–
–
–
–
–
–
–
0.2
–
0.1
–
0.9
–
–
0.8
–
–
–
–
–
tr
–
0.1
1.3
–
0.3
–
–
0.1
1.8
–
–
–
0.9
–
0.2
1.1
–
–
–
0.8
–
0.4
–
–
tr
–
–
1.3
0.1
–
–
–
–
–
–
–
–
0.2
–
–
–
–
–
–
–
–
–
–
–
–
–
0.2
–
–
–
3.6
–
0.7
0.5
6.2
–
tr
–
–
–
3.8
–
–
–
–
0.3
0.2
–
–
–
0.7
tr
7.6
–
–
–
0.7
–
–
–
–
–
–
4.2
–
2.0
–
–
–
tr
0.7
7.1
0.1
–
–
tr
–
–
0.2
–
–
0.7
–
–
0.3
–
–
–
–
–
A
A
A
A
A
A
A
A
A
0.2
–
0.1
–
–
–
3.5
tr
–
0.2
–
0.2
0.1
–
–
–
–
1.1
–
–
–
–
–
0.2
0.4
–
0.2
–
–
0.1
0.1
0.3
–
–
–
–
–
0.1
–
–
9.3
–
0.2
0.2
–
–
0.7
–
1.3
0.3
–
–
–
0.1
0.1
2.9
–
0.2
–
–
2.5
–
0.4
–
0.3
–
0.1
0.3
1.1
–
0.2
–
0.1
–
0.1
–
–
2.4
0.1
–
–
–
–
–
–
–
3.4
0.3
–
–
–
–
0.3
–
2.9
0.1
–
–
–
–
0.1
–
–
0.1
0.1
–
0.3
–
0.2
tr
–
tr
–
2.6
0.1
0.2
0.2
–
0.2
0.1
tr
–
5.5
–
0.2
–
0.1
–
–
–
–
–
–
–
–
1.9
5.3
–
–
–
0.5
–
–
–
0.6
tr
A
B
A
A
A
B
A
A,B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A,B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
(continued on next page)
543
V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
Table 2 (continued)
Essential Oil Components
RI(obs)
Aaca
Chen-1
Chen-2
Cpan
Lrig
Lsca
Mten
Scap
Targ
Tpar
ID Methoda
a-cadimene
1541
1561
1566
1581
1600
1603
1607
1609
1612
1625
1631
1632
1653
1642
1644
1646
1646
1656
1668
1674
1687
1686
1724
1741
1760
1922
1962
2132
2138
2140
–
–
–
–
–
–
0.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.1
–
–
–
w97
–
–
tr
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
tr
–
–
–
tr
–
–
–
w99
–
–
0.8
–
–
–
–
–
–
–
–
–
–
–
–
–
tr
–
–
–
–
–
tr
–
–
–
0.1
–
tr
tr
w98
0.1
3.3
–
–
–
–
–
–
–
–
0.1
–
–
–
–
1.6
–
–
–
–
–
–
–
tr
–
–
–
–
–
–
w92
–
–
–
1.5
–
–
0.3
–
–
–
–
–
–
0.3
–
–
–
–
–
–
–
–
–
–
–
–
0.9
–
10.9
–
w90
0.1
0.4
–
–
–
–
–
0.1
–
–
–
–
–
–
–
2.0
–
3.6
–
–
–
–
–
0.4
–
–
–
–
–
–
w90
0.1
–
–
–
–
–
–
0.2
–
–
0.2
–
–
–
1.9
0.3
–
3.7
–
–
–
0.5
–
tr
0.2
–
3.9
–
–
–
w84
–
1.1
–
–
0.2
–
1.0
–
0.2
–
–
0.1
–
–
–
–
–
–
0.3
0.5
–
–
–
–
–
0.5
1.5
0.6
0.2
0.6
w91
–
0.3
–
–
–
–
0.6
–
–
–
–
–
1.1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
w88
tr
0.1
–
0.5
0.1
0.2
–
–
–
0.1
–
–
–
–
0.2
0.1
–
–
–
–
0.5
–
–
0.5
tr
–
0.6
0.4
3.0
–
w97
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A,B
A
A
A
B
B
A,B
germacrene B
(E)-nerolidol
spathulenol
guaiol
5-epi-7-epi-a-eudesmol
geranyl isovalerate
b-oplapenone
tetradecanal
10-epi-g-eudesmol
1-epi-cubenol
g–eudesmol
pogostol
6,6-dimethyl-6(3-methylphenyl)-heptan-3-one
epi-g-cadinol
a-muurolol
3-butylphthalide
a-cadinol
trans-calaminen-10-ol
tetradecanol
a-bisabolol
eudesm-4(15),7-dien-1b-ol
senkyunolide
mint sulfide
benzyl benzoate
methyl hexadecanoate
hexadecanoic acid
linoleic acid
osthole
linolenic acid
% of oil identified
a
A ¼ observed RI and mass spectra correlated with those available in (Adams, 2007); B ¼ observed retention times and mass spectra matched with those
of authentic samples; C ¼ observed RI and mass spectra matched with those in Kollmannsberger et al., 1998.
species group, the Black Swallowtail butterfly (Papilio polyxenes), has been thoroughly studied to determine the specific
chemical constituents involved in female attraction. Feeny et al. (1989) and Baur et al. (1993) found that host-searching
behavior and alighting on leaves increased in the presence of certain volatile components of carrot oil (Daucus carota), but
these compounds alone did not enhance oviposition rates. This is because upon alighting, female Papilionids drum leaf
surfaces with foretarsi to “taste” for certain non-volatile chemicals before depositing eggs (Feeny et al., 1983, 1988). Electroantennogram (EAG) studies with P. polyxenes females showed that both non-polar and polar fractions of the carrot oil
produced EAG spikes, but polar fractions produced greater spikes than the considerably more prevalent non-polar fractions
(Baur et al., 1993; Feeny et al., 1989), indicating that polyxenes females may rely more on the polar components when
searching for host plants. However terpinolene and myrcene, two non-polar components, produced good EAG spikes. They
found that landing frequency was greater on artificial leaves soaked with the four polar compounds sabinene hydrate, (Z)-3hexenyl acetate, 4-terpineol, and bornyl acetate, than on artificial leaves soaked with the more abundant non-polar fractions
(Baur et al., 1993). None of these four polar compounds were present across the nine P. indra host plants. Two of them, 4terpineol and bornyl acetate were present in only six of the nine species, while sabinene hydrate and (Z)-3-hexenyl acetate
were entirely absent (Table 2). Our results for P. indra host plants revealed that the non-polar pinenes (a and b), and bphellandrene þ limonene were consistently present and frequently in highest proportions across the nine host species (Table
2). These compounds emit strong citrusy/piney aromas characteristic of these plants. Combined pinene composition ranged
from 1.0% (C. hendersonii) to 49.1% (Aletes acaulis), and b-phellandrene þ limonene composition ranged from 2.7% (C. hendersonii) to 39.4% (Tauschia parhshii). Myrcene, p-cymene, the b-ocimenes, terpinene, g-terpinolene, and the polar compound
a-terpineol were also present in all nine host plants (Table 2) and contribute to the citrusy/piney aromas of the host plants.
The results therefore provide evidence that different members of the Papilio machaon species group, thought closely related,
do not always utilize the same volatile essential oil components for host plant orientation. P. indra females may use the
prevalent non-polar constituent of the oils, instead of the polar constituents. We suspect that Indra Swallowtail females are
attracted to appropriate hosts by the aromatic qualities of one compound, or a few of these compounds acting in synergy. The
host shift from the ancestral family Rutaceae to the Apiaceae was likely facilitated by the prominent citrus/piney aromas
emitted by these volatile compounds (Dethier, 1941; Jermy, 1984).
The two C. hendersonii samples contained the lowest percentages of the pinenes and the b-phellandrene þ limonene.
Methyl chavicol and (E)-anethole, together contributed as much as 63.8% of the oil (Table 2) and together provide the anise
aroma of this species. Dethier (1941) found that methyl chavicol was the best attractant for P. polyxenes. However his study
involved caterpillars instead of adults. Based on Dethier’s results and the high percent composition of these two compounds,
544
V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
Fig. 1. Cluster tree diagram from the first six PCA loadings based on the compositions of the essential oils of P. indra host plants and non-host plants.
one or both are potential attractants for Indra Swallowtails. Dethier inferred that for a given Apiaceae host the most
prominent compounds of the oil were likely used as key olfactory attractants, and to human olfactory senses these
compounds represent the characteristic aroma of the plant. We theorize that this is true for P. indra. Eight of the nine P. indra
hosts we studied have citrusy or pine-like aromas, the exception being C. hendersonii, and amongst the essential oil
components of the nine host plant, the pines and limonene are suspected to be attractants.
Percent concentrations of essential oil components may vary geographically, but the essential oil biosynthetic pathways
are expected to be similar as seen for C. hendersonii (Table 2, Fig. 1). Indra Swallowtails are known to use a particular host
species across the host’s geographical range (pers. obs., W.H. Whaley) and are likely using the same chemical attractants
produced by these pathways.
4.2. Host and non-host essential oil compositions: a comparison through PCA
PCA and cluster analysis on the oil compositions of the indra host species and the non-host species, Lomatium dissectum, L.
dasycarpum, L. utriculatum, and L. nevadense, indicated that the oil compositions of the non-hosts fall outside of the oil
compositions of the nine hosts (Table 3 and Fig. 1). These non-host species do not emit the citrusy/piney aromas, or the anise
aroma (C. hendersonii) of most indra hosts (pers. obs., W.H. Whaley). Twelve essential oil components (bold type, Table 3)
were the most important separators. Of these citronellol, citronellal, a- and b-pinenes, b-phellandrene þ limonene, and
methyl chavicol were important separators due to presence in all or most of the host plants, while being absence and/or in
low concentrations in the non-host plants (Table 3). Individual compounds or synergistic combination of these may act as
female attractants. Certain compounds in the four non-host species, e.g., (Z)-3-hexenol (Table 3), may act as deterrents to
oviposition. However non-use of a plant species by an insect, even though its larvae could feed successfully on it, may be due
to lack of appropriate olfactory attractants so that females never make physical contact with the plant. For example, lepidopterists regularly use a non-host species for successful laboratory rearing (pers. obs., W.H. Whaley).
Might shared secondary plant metabolites be good indicators of shared ancestry for the Apiaceae? Cluster analysis on PCA
loadings of essential oil components produced results contrary to what was expected since the Lomatium, Cymopterus and
545
V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
Table 3
Aletes acaulis (Aaca), Cymopterus hendersonii (Chen-1, and Chen-2), Cymopterus panamintensis (Cpan), Lomatium rigidum (Lrig), Lomatium scabrum (Lsca), Musineon
tenuifolium (Mten), Sphenosciadium capitellatum (Scap), Tauschia arguta (Targ), Tauschia parishii (Tpar), Lomatium dissectum (Ldis), Lomatium dasycarpum (Ldas),
Lomatium utriculatum (Lutr), and Lomatium nevadense (Lnev) essential oil components of interest and their compositions. The first 12 components (bold type)
are arranged from highest to lowest PCA loading and represent those which best separate P. indra host plants from the four non-host plants. The other listed
components are frequently discussed in the text.
Components
Aaca
Chen-1
Chen
(Z)-3-hexenol
hexanol
furfural
benzaldehyde
o-guaiacol
2-phenylethyl alcohol
citronellol
citronellal
a-pinene
b-pinene
b-phellandrene D limonene
methyl chavicol
myrcene
(Z)-b-ocimene
(E)-b-ocimene
g-terpinene
terpinolene
linalool
a-terpineol
4-terpineol
bornyl acetate
(E)-anethole
% partial composition
–
–
–
–
–
–
0.3
–
10.8
38.3
29.0
0.2
8.1
1.5
0.5
0.3
0.3
0.5
1.1
0.5
–
0.04
91.4
–
–
–
–
–
–
2.7
1.2
0.3
0.7
2.7
29.9
2.1
0.2
3.1
1.8
0.2
1.2
0.7
0.8
–
11.8
59.4
–
–
–
0.04
–
–
3.5
0.9
0.6
1.2
4.7
42.3
1.6
0.2
2.9
0.5
0.04
–
0.6
0.6
–
21.5
81.14
a
b
c
2
Cpan
Lsca
Lrig
Tpar
Mten
Scap
Targ
Ldisa
Ldasb
Lutrb
Lnevc
–
–
–
–
–
–
1.6
0.9
0.9
0.9
19.8
0.2
30.0
4.9
4.5
0.04
0.2
3.6
0.2
0.3
0.6
–
68.64
–
–
–
–
–
–
0.4
0.1
1.4
4.6
26
–
8.2
11.8
4.0
0.3
0.8
–
0.5
–
3.7
–
61.8
–
–
–
–
–
–
0.5
0.1
6.9
3.5
28.6
0.8
3.9
0.2
0.6
0.1
0.8
0.8
0.1
0.2
2.4
–
49.5
–
–
–
–
–
–
0.5
0.1
6.4
8.8
39.4
0.1
2.6
1.3
1.5
1.2
0.8
–
0.1
0.1
0.7
–
63.6
–
–
–
–
–
–
2.4
0.4
0.6
0.1
25.5
–
1.0
0.3
0.4
0.3
0.2
2.3
0.4
0.4
1.1
–
35.4
–
–
–
–
–
–
–
–
1.0
0.04
11.3
–
0.2
17.9
4.3
6.4
1.2
1.2
0.3
–
–
–
43.84
–
–
–
–
–
–
–
–
0.5
2.4
6.4
–
3.0
13.1
5.7
0.2
9.2
–
0.3
–
0.2
–
41
18.5
1.0
0.4
0.3
0.1
0.2
–
–
–
–
–
–
6.0
0.2
1.0
–
–
0.6
0.2
0.1
–
–
28.3
1.8
–
1.0
–
–
–
–
–
–
–
–
–
0.1
0.3
0.1
0.4
–
0.5
0.8
1.7
0.6
–
7.3
0.5
0.2
–
–
0.6
0.3
–
–
0.1
0.1
0.2
–
0.1
–
0.2
0.3
0.1
0.4
0.6
11.2
–
–
14
3.9
0.6
0.4
0.1
–
–
–
–
0.2
0.1
4.0
–
1.6
0.8
5.1
3.3
1.7
0.4
0.2
0.8
–
–
23.2
Bairamian et al., 2004.
Asuming et al., 2005.
Beauchamp et al., 2007.
Tauschia species were not closely clustered. However as expected the two C. hendersonii samples clustered nicely (Table 3 and
Fig. 1). A phylogenetic analysis using nuclear rDNA internal transcribed spacers (ITS) on 85 taxa of Apiaceae (Downie et al.,
2002), included ten of our species and clustered L. rigidum and T. parishii together in a similar manner as represented in
Fig. 1. These two indra host species are phenotypically very similar and often confused when encountered in the field. Their
similarity in essential oil composition and morphology may indicate a closer relationship than is presently represented by
their placement in separate genera. To infer true relationships from our data would be tenuous, because phylogenies for
family Apiaceae and specifically for genera Cymopterus, Lomatium, Aletes, Tauschia and Musineum, are in great flux (Downie
et al., 2002; Soltis and Novak, 1997). Because phylogenies for the Apiaceae using morphological and molecular data are not in
agreement, it is difficult to make comparisons with those based on secondary metabolites. However, we tend to concur with
the molecular (ITS) data because the methodology involves presumed neutral characters.
4.3. Machaon species group and citrus family connection in southwestern U.S.
Several taxa in Rutaceae have essential oil compositions (De Pasquale et al., 2006; Kirbaslar and Kirbaslar, 2004; Lota et al.,
2001; Ruberto et al., 1994, 1997; Verzera et al., 2005) that are remarkably similar to the essential oil components of Indra
Swallowtail host plants. This may account for the observation that in the southern deserts of California P. indra fordi females
are occasionally attracted to the Rutaceae species Thamnosma montana (pers. obs., W.H. Whaley). The result is attraction with
no apparent oviposition, a likely result of contact chemical “tasting” during foretarsi drumming of leaves. In these desert
mountains another machaon member, the Desert Black Swallowtail butterfly (P. polyxenes coloro) uses T. montana as its main
host. Not unexpectedly, b-phellandrene and limonene comprise as much as 64% of the essential oils of this species (Tucker
et al., 2005) and its leaves and stems emit strong lemony aromas. In the same regions P. p. coloro occasionally oviposits on
the P. i. fordi host plant C. panamintensis (pers. obs., W.H. Whaley) and produces viable offspring. Interestingly, during
infrequent population explosions of P. indra fordi, the caterpillars upon decimating their usual larval host C. panamintensis,
will crawl to and feed on the nearby T. montana, the usual host of P. p. coloro (pers. obs., W.H. Whaley). It is not known if these
T. montana-fed indra caterpillars survive to produce viable, fertile adults. It appears that certain citrusy essential oil
components attract the indra caterpillars to T. montana, but the female Indra Swallowtails are not prone to oviposit on it,
possibly because of contact chemical deterrents in the leaves.
Another member of the machaon species group, P.zelicaon also used host plants in Apiaceae. Within the last 100 years P.
zelicaon has begun to use orchard Citrus in regions of the southwest (Emmel and Shields, 1978; Shapiro and Masuda, 1980),
again hinting that family Rutaceae is the ancestral host family for Papilio. The small number of Papilio which uses apiaceous
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V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547
plants, e.g., the eight members of the machaon species group, suggests a very recent host shift from the Rutaceae, perhaps as
early as the early Pleistocene, (Berenbaum, 1995; Sperling, 1987; Sperling and Feeny, 1995).
4.4. Summary and need for further study
Several compounds in P. indra larval host plants emit citrusy/piney aromas, prominent ones being b-phellandrene,
limonene the two pinenes, citronellol, citronellal and g-terpinene, components well documented in the Rutaceae, and these
aromatic compounds are possibly important constituents involved in the attraction. Since Rutaceae is the primary host family
and the probable ancestral host family of Papilio, these essential oil constituents provide an excellent roadmap for further
study. Of the P. machaon species group members only P. polyxenes has been studied to determine the volatiles that attract
females to their host plants. Whether the predominant non-polar essential oil components or the less common polar
components are key attractants is a matter to be resolved using laboratory experiments with live females and purified
essential oil compounds. Better understanding of the chemically stimulated host plant search behavior of P. indra, a species
holding considerable interest amongst butterfly enthusiasts, may shed light on the evolution of this behavior amongst the
other members of the P. machaon species complex.
Acknowledgements
Partial funding to W.H. Whaley by a Scholarly Activities Grant at UtahValley University is gratefully acknowledged. The
authors wish to thank Larry Blakely, John Emmel, Kenneth Davenport, Dave Wikle, Paul Rumpa and Grace Kostel for collecting
some of the plants. Thanks to Floyd Howell, Staff Emeritus of the Chemistry Department, California State Polytechnic
University, Pomona, for assistance in the maintenance of the instruments.
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