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 UnitedStates 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

aDepartment of Chemistry, California State Polytechnic University, Pomona, CA 91768, USAbDepartment of Biology, College of Science and Health, Utah Valley University, 800 W. University Parkway, Orem, UT 84058, USAcDepartment of Statistics, Brigham Young University, Provo, UT 84662, USA

a r t i c l e i n f o

Article history:Received 15 September 2009Accepted 29 May 2010

Keywords:ApiaceaeEssential oil compositionIndra Swallowtail butterflyPapilio indraAletesCymopterusLomatiumMusineonSphenosciadiumTauschia

* Corresponding author. Tel.: þ1 801 863 8607; faE-mail address: wwhaley@uvu.edu (W.H. Whale

0305-1978/$ – see front matter � 2010 Elsevier Ltddoi:10.1016/j.bse.2010.05.010

a b s t r a c t

Aletes acaulis, Cymopterus hendersonii, Cymopterus panamintensis var. acutifolius, Lomatiumrigidum, Lomatium scabrum var. tripinnatum, Musineon tenuifolium, Sphenosciadium cap-itellatum, Tauschia arguta and Tauschia parishii are among the twenty-two species of theApiaceae family to which female Indra Swallowtail butterflies (Papilio indra: Lepidoptera)are attracted for oviposition. Because plant volatile oils are known to be attractants forfemale butterflies, the percent composition of the essential oils of each species wasstudied. Amongst the nine host plants 168 essential oil components were identified rep-resenting between 84% and 99% of the oils. Principal Components Analysis and hierarchicalcluster analysis on the essential oil compositions of the larval host plants against four non-larval host plants separated the hosts from the non-hosts into distinct clusters. Volatilecomponents of the oils common to the nine species of Apiaceae are correlated with theexpression of physiological attraction behavior by the butterfly.

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1. Introduction

Butterflies in the genus Papilio (Lepidoptera: Papilionidae) are known to be attracted to an appropriate larval host plant byolfactory 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 vola-tiles for orientation to appropriate host plants. The Indra Swallowtail butterfly (Papilio indra) has been observed to oviposit ontwenty-two species in the family Apiaceae (>275 species inwestern U.S.; Cronquist et al., 1997; Hickman,1993; Hitchcock andCronquist, 1973; Kearney et al., 1960). The butterfly is found in widely scattered populations in mountainous regions acrossthe western U.S. and generally one, rarely two, of these host plants are utilized by any one population (Whaley, 2000). Atseveral locations in the Great Basin andMojave Desert mountain ranges its larval host plants grow as widely scattered singlestucked within rock crevices to conserve moisture, which often makes them difficult for researchers to find (e.g., Cymopteruspanamintensis). However, the female’s sensitive antennae sensilla allow her to find host plants with ease. The butterfly’sattraction to its host plants has been ascribed to the citrusy/piney aroma of these plants, with just three emitting anise-like orcelery-like aromas (Dev et al., 2007; Whaley, 2000). It is conceivable that other aromatic as well as non-aromatic componentsmight also be responsible for the butterfly’s behavior. However, whatever attracts the butterfly to the plant in the first place, it

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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 toovipositing has been observed to involve compounds other than essential oil components (Feeny et al., 1988). Our assumptionof the involvement of certain components of the essential oils of the plants in attracting the butterfly has lead us to the studyof the composition of these oils in its Apiaceae larval host plants (Beauchamp et al., 2009; Dev et al., 2007) anticipating someconsistencies in oil components across the hosts. In the present communication we report the essential oil compositions ofnine 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 transportationto the laboratory. They were deposited at the indicated herbaria and their accession numbers assigned. Specimens werecollected in the flowering or seed stages, either of which is known to be used for oviposition by Indra Swallowtail butterfly.

Aletes acauliswas 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 theiressential 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. acutifoliuswas collected off Rabbit Spring Road near where Highway 18 ends and Highway247 starts, Granite Mountains, San Bernardono Co., California; GPS coordinates lat. 34� 28

0N, long. 116� 590 W, elev. 3219 ft.

(access. no. UVSC 13100).Lomatium rigidumwas collected in Bishop Canyon along Highway168 at1.3miles below South Lake Road junction, Inyo Co.,

California, 37�1603000 N, 118� 3403200 W, elev. 7415 ft. (access. no. RSA 745006).Lomatium scabrum var. tripinnatumwas collected along highway18 at 1.3 miles north of St. George, Washington Co., Utah,

37� 80 44.8600N, 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�

03031.5200 N, 103� 21051.3900 W, elev. 4197 ft. (access. no. UVSC 13106).Sphenosciadium capitellatumwas 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., Cal-ifornia, 33� 410 4400 N, 116� 450 3900 W, 4000 ft elev. (access. no. UVSC 13102).

Tauschia parishiiwas collected at Butterbredt Peak, Jawbone Canyon Road, Kern Co., California, GPS coordinates 35� 23004

00

N, 118� 09022

00W, 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 therespective 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)wasused for determining composition and Kováts retention indices (RI). The oven temperature was programmed at 50 �C for10 min and then 3 �C/min to 240 �C, after which it was maintained at this temperature for 5 min. The GC/MS analyses wereconducted with an AGILENT 5973 Network Mass Selective Detector interfaced with AGILENT 6950 GC system fitted witha capillary column matching the one used with the HP 6890 GC. The GC of the GC/MS was programmed at 50 �C for 10 minfollowed 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 theessential oil compositions of the nine P. indra host plants presented here and the previously published essential oilcompositions 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 theyare prevalent within the butterfly’s habitat, and because genus Lomatium is more commonly used by the butterfly (14 of the22 host species). The first two PCA loadings were used to determine the important essential oil compounds separating hostfrom non-host. Then a hierarchical cluster analysis was performed on the first six PCA loadings to group the plants intopotential phylogeny.

V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547540

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 oilcomponents 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 oilcomponents was established by an excellent match of both their RI as well as mass spectrawith the RI andmass spectra of thecorresponding compounds available in the literature as listed under Identification Methods. Among the nine host species 168compounds were identified. The percentage of the identified components varied from>84% forM. 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 aromawas due to the b-phellandrene þ limonene and the pinene (Table 1). For the two samples of C. hendersonii, methyl chavicol and (E)-anetholeproduce its anise aroma. When evaluating each plant’s aroma it was sometimes difficult to distinguish a citrusy aroma froma 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 similarcompounds, only themost prevalent essential oil components were used for the statistical analysis. Thesewere determined tobe 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-hostplants. Eighty three of the168 compounds in the host plant and non- host plant essential oils met this criterion. We foundthrough preliminary principal component analysis that if there was a large discrepancy in concentration levels of an essentialoil compound used in our analysis, for example 30%–0.2% for myrcene, that those plants showing small amounts were treatedas if they were effectively the same as zero. This did not seem to be the correct way of handling the large discrepancies inconcentrations and did not support the objectives. Therefore, we felt that using presence or absence would be morerepresentative of the makeup of each plant. Thus, PCA on the presence or absence of these 83 compounds was used todistinguish 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 treeproduced by using these principal components revealed that the essential oil compositions of the four non-host speciesdiffered significantly (distance >1.00) from the host species (Fig. 1). The C. hendersonii samples from Montana and Utahpopulations were most similar in essential oil constituents. However, the Lomatium species and the two Tauschia speciesdiffered 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 fewrelated plant families. A majority of the 200 þ species of Papilio use plants in the Rutaceae (citrus family), the familyconsidered ancestral to the genus (Scriber, 1973), but eight species comprising what is referred to as the Papilio machaonspecies group, which includes P. indra, use members of the Apiaceae (the umbels) as larval hosts. In the papilionids attractionto 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 onemember of themachaon

Table 1Yields of oils from Aletes acaulis (Aaca),Cymopterus hendersonii (Chen-1 and Chen-2),Cymopterus panamintensis (Cpan), Lomatium rigidum (Lrig), Lomatium sca-brum var. tripinnatum (Lsca), Musineon tenuifolium (Mten), Sphenosciadium capitellatum (Scap),Tauschia arguta (Targ), and Tauschia parishii (Tpar).

Plant name g plant mg oil % oil

Aaca 104 129 0.12Chen-1 77 79 0.10Chen-2 65 1066 1.64Cpan 50 254 0.51Lrig 73 68 0.09Lsca 273 430 0.16Mten 225 63 0.03Scap 173 156 0.09Targ 180 489 0.27Tpar 480 756 0.16

Table 2Composition 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 854 – – tr 0.8 – – – – – – C(E)-2-hexenal 856 – – – 0.8 tr – 0.1 tr – 0.1 Aisobutyl isobutyrate 921 – 0.8 tr – – – – – – – Ca–thujene 932 0.2 0.2 0.1 0.1 0.1 – 0.1 0.1 – 0.3 Aa–pinene 938 10.8 0.3 0.6 0.9 6.9 1.4 0.6 1.0 0.5 6.4 A,Bcamphene 953 0.2 0.1 tr 0.1 0.9 0.1 0.1 – – 0.6 Abenzaldehyde 963 – – 0.04 – – – – – – – A,Bsabinene 978 – 11.3 7.8 0.6 0.1 – 0.4 0.2 0.1 0.4 Ab-pinene 979 38.3 0.7 1.2 0.9 3.5 4.6 0.1 tr 2.4 8.8 A,Bmyrcene 993 8.1 2.1 1.6 30.0 3.9 8.2 1.0 0.2 3.0 2.6 A,Ba–phellandrene 1002 0.1 tr tr 0.2 0.6 0.7 0.1 5.0 1.0 2.0 Aisobutyl 2-methylbutyrate 1005 – 0.2 tr – 0.1 0.5 tr – – – Bisobutyl 3-methylbutyrate 1009 – 0.6 0.1 – – – – – – Bd-3-carene 1010 – – – – – 0.6 – 0.6 1.6 – A(E)-2-hexenyl acetate 1011 – – – – – 0.2 – – – – Aisobutyl isovalerrate 1014 – – – – – – – 0.5 – – Aa–terpenene 1017 0.1 0.6 0.2 tr – – – 0.1 0.3 0.1 A2-methylbutyl isobutyrate 1021 – 0.2 – – – – – – – – Cp-cymene 1027 0.2 0.6 tr 0.2 0.9 0.3 0.1 2.7 0.4 0.1 A,Bb-phellandrene þ limonene 1031 29.0 2.7 4.7 19.8 28.6 26.0 25.5 11.3 6.4 39.4 A,B1,8-cineole 1034 0.1 0.1 0.1 – – – – – – – A,B(Z)-b-ocimene 1043 1.5 0.2 0.2 4.9 0.2 11.8 0.3 17.9 13.1 1.3 Abutyl 2-methylbutyrate 1048 – tr tr – – 0.4 – – – – B(E)-b–ocimene 1054 0.5 3.1 2.9 4.5 0.6 4.0 0.4 4.3 5.7 1.5 Aisobutyl angelate 1058 – – – – 0.2 – – – – – Bg–terpinene 1062 0.3 1.8 0.5 tr 0.1 0.3 0.3 6.4 0.2 1.2 Acis-sabinene hydrate 1070 – 0.1 – – – – – – – – Am-cresol 1076 – – – – – – 0.1 – 0.2 – Acis-linalool oxide (furanoid) 1076 – – – 0.1 – – – – – – Abutyl angelate 1083 – – – – 0.3 – tr – – – Bterpinolene 1088 0.3 0.2 tr 0.2 0.8 0.8 0.2 1.2 9.2 0.8 A6,7-epoxymyrcene 1094 – – – 0.1 – – – – – – A6-camphenone 1096 – – – – 0.3 – – – – – Alinalool 1100 0.5 1.2 – 3.6 0.8 – 2.3 1.2 – – A,B3-methylbutyl 2- methylbutanoate 1102 tr – – – tr – 0.3 tr – 0.3 A,B2-methylbutyl 2- methylbutanoate 1106 – – tr – 1.1 1.5 0.1 0.6 – 0.6 A.B3-methylbutyl 3- methylbutanoate 1107 – 0.3 0.1 – – – tr 2.0 – 0.1 B2-methylbutyl 3-methylbutanoate 1112 0.1 – 0.1 – 0.1 – – 3.8 – 0.2 Bdehydrosabina ketone 1122 – 0.1 0.1 0.1 0.2 0.1 – – – 0.1 Aallo-ocimene 1132 tr – – 0.1 – 0.1 – 0.3 – tr Acamphor 1146 – – – 0.3 0.4 – – – – 0.1 A,B3-methyl-2-buten-1-yl 2-methylbutyrate 1146 – – – – – – 0.2 – – – Bhexyl isobutyrate 1153 tr 0.3 – – – – – – – – Aisoamyl angelate 1154 – – – – 0.2 – – – – – Bcitronellal 1155 – 1.2 0.9 0.9 0.1 0.1 0.4 – – – A2-methylbutyl angelate 1158 – – – – 0.9 – – – – 0.5 Bborneol 1167 tr – – – 0.1 – – – – – Alavandulol 1171 – – – 0.1 – – – 0.3 – – A4-terpineol 1176 0.5 0.8 0.6 0.3 0.2 – 0.4 – – 0.1 Aviridine 1178 – – – – – – – 0.4 – – Anaphthalene 1181 – – – – – – – 0.2 – Ap-cymenene-8-ol 1186 – – – – – – – – 0.7 – Acryptone 1188 0.1 – – 0.4 5.6 0.3 0.5 – – 0.1 Aamyl angelate 1190 – – – – 0.3 – – 0.2 – 0.4 Ba-terpineol 1192 1.1 0.7 0.6 0.2 0.1 0.5 0.4 0.3 0.3 0.1 A(E)-4-decenal 1194 – – – – – – 0.9 – – – A(Z)-undecenal 1195 – – – 0.5 – – – – – – Amethyl chavicol 1196 0.2 29.9 42.3 0.2 0.8 – – – – 0.1 A2-methylbutyl tiglate 1200 – – – 0.3 – – – – – – B4-methylpentyl 2-methylbutyrate 1201 – – – – 0.4 – – – – – Bg–terpineol 1202 – – 0.7 – – – – – – – Adecanal 1207 – – – 0.6 – – 1.2 – – tr Acitronellol 1228 0.3 2.7 3.5 1.6 0.5 0.4 2.4 – – 0.5 Athymol methyl ether 1236 tr 0.4 0.1 – – – – – – – Aneral 1241 – tr 0.2 – – – – – – – Acuminal 1242 – – – – 0.5 – – – – 0.1 Acarvacrol methyl ether þ hexyl 3-methylbutyrate 1247 – 0.4 tr – – – – – – Acarvone 1247 – – – – 0.1 – – – – – Apiperitone 1254 – 0.3 0.3 – 0.1 – – – – – A

V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547 541

Table 2 (continued)

Essential Oil Components RI(obs) Aaca Chen-1 Chen-2 Cpan Lrig Lsca Mten Scap Targ Tpar ID Methoda

geraneol 1258 – – – – – 0.5 1.5 – – – Alinalool acetate 1258 – – – 1.2 – – – – – – Amethyl citronellate 1264 – – – – 0.1 – – – – tr Ageranial 1274 – – 0.3 – – – – – – – Acitronelly formate 1275 – – – – – – 0.2 – – – Adihydrolinalool acetate 1277 – – – 0.1 0.3 – – – – 0.2 Ap-menth-1-en-7-al 1277 – – – – – 0.1 – – – – Aa-terminen-7-al 1283 – – – – 0.1 – – – – – Abornyl acetate 1288 – – – 0.6 2.4 3.7 1.1 – 0.2 0.7 AC6H11-angelate/tiglate 1289 – – – – 0.2 – – – – –

(E)-anethole 1290 tr 11.8 21.5 – – – – – – A(Z,Z,Z)-3,6,9-tridecatriene 1291 – – – – 2.0 – – – – 0.3 Blavandulyl acetate 1294 – – – – – 0.6 9.3 1.0 – – Acarvacrol 1300 – – – – – – – 0.3 – – Aundecanal 1306 – – – – – – 0.2 – – – A4-methylhexyl 2-methylbutanoate 1307 – – – – 0.2 – 0.1 – – Bneoiso-pulegyl acetate 1314 – – – – – – 0.2 – – – Ap-vinyl guaiacol 1314 – tr tr – – – – – – A,B(E,E)-2,4-decadienal 1322 – – 0.1 tr – – – – – – Amyrtenyl acetate 1334 – – – – – – 0.7 – – – A3-oxo-p-menth-1-en-7-al 1336 – – – 0.1 0.8 – – – – – Atrans-carvyl acetate 1339 – – – – – – 1.3 – – – Aa-cubebene 1348 – – – – – – 0.3 – 0.2 0.1 Aeugenol 1360 – tr 0.2 – – – – – – – Acyclosativene 1370 – – – – 0.1 – – – – – Alongicyclene 1372 – – – – – – – – – 0.1 Aa-copaene 1376 – – – – 0.1 0.1 0.1 0.2 3.6 0.1 Adaucene 1380 – – – – – – 0.1 – – – Ab-bourbonene 1388 0.1 – – 0.6 0.4 0.5 2.9 0.1 0.7 0.3 Ab-cubebene 1388 – – – – – – – 0.5 – Ab-elemene 1393 – – – 0.5 0.2 1.0 0.2 0.9 6.2 0.2 Alongifolene 1406 – – – – – – – – – tr Amethyl eugenol 1407 0.1 19.0 4.3 – 0.1 – – – tr Adodecanal 1409 – – – – – – 2.5 0.8 – – Aa-gurjunene 1412 – – – – – – – – – tr Ab-ylangene 1419 – – – – – – 0.4 – – – Ab–caryophyllene 1422 tr 0.2 0.1 – 3.5 1.2 – – 3.8 2.6 A,Bb-copaene 1432 – – – 0.1 tr 0.1 0.3 – – 0.1 Aa-guaiene 1438 – – – – – – – – – 0.2 A2-methylbutyl benzoate 1442 – – – – 0.2 – 0.1 tr – 0.2 Aaromadendrene 1448 – – – – – – 0.3 – – – Aa–humulene 1453 – – tr 1.2 0.2 0.4 1.1 0.1 0.3 0.2 A(E)-b–farnesene 1459 tr – tr – 0.1 – – 1.3 0.2 0.1 Acis-muurola-4(14),5-diene 1468 – – – – – – 0.2 – – tr A4,5-diepi-aristolochene 1474 – – – – – – – 0.3 – – Atrans-cadina-1(6),4-diene 1477 – – tr tr – – 0.1 – – 5.5 Ab-chamigrene 1477 – – – – – – – – 0.7 – Ag-muurolene 1479 1.2 – – 0.2 1.1 0.3 0.1 0.1 tr 0.2 Aamorpha-4,7(11)-diene 1481 – – – – – – – 1.8 7.6 – Aar-curcumene 1484 – – – – – – – – – 0.1 Agermacrene D 1484 – – – 3.5 – 2.1 2.4 – – – Acitronellyl isobutyrate 1484 0.3 0.1 tr – – – 0.1 – – – Ab-selinene 1488 – – – 0.2 – – – 0.9 0.7 – A(E)- b-ionone 1489 – – – – 0.2 – – – – – A2-phenylethyl 3-methylbutanoate 1493 – tr – – 0.4 – – 0.2 – – Aa-selinene 1496 – – – – – – – 1.1 – – Avalencene þ benzyl tiglate 1496 – – – – 0.2 – – – – – Aa–zingiberine 1496 – – 0.2 1.2 – – – – – – Aviridiflorene 1497 – – – – – – – – – 1.9 Abicyclogermacrene 1498 1.0 6.1 – – 0.1 3.0 3.4 0.8 4.2 5.3 Aa-muurolene 1500 – – – 0.2 0.1 0.5 0.3 – – – Agermacrene A 1502 – – – 0.4 0.3 0.3 – 0.4 2.0 – A(E,E)-a–farnesene 1509 0.2 0.7 0.1 – – – – – – – Aa-cuparene þ (E,E)-a- farnesene 1510 – – – – – – – – – 0.5 Alavandulyl 2-methylbutyrate 1512 – – – – – – – tr – – Ag-cadinene 1516 – – – 0.3 – 0.5 0.3 – tr – A(Z)-g-bisabolene 1516 – – – – – – – – 0.7 – Ad-cadinene 1526 0.1 – – 2.6 0.1 3.2 2.9 1.3 7.1 0.6 Atrans-cadina-1(2),4-diene 1534 – – – – – 0.1 0.1 0.1 0.1 tr A

(continued on next page)

V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547542

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 – – – 0.1 – 0.1 0.1 – – tr Agermacrene B 1561 – – – 3.3 – 0.4 – 1.1 0.3 0.1 A(E)-nerolidol 1566 – tr 0.8 – – – – – – – Aspathulenol 1581 – – – – 1.5 – – – – 0.5 Aguaiol 1600 – – – – – – – 0.2 – 0.1 A5-epi-7-epi-a-eudesmol 1603 – – – – – – – – – 0.2 Ageranyl isovalerate 1607 0.3 – – – 0.3 – – 1.0 0.6 – Ab-oplapenone 1609 – – – – – 0.1 0.2 – – – Atetradecanal 1612 – – – – – – – 0.2 – – A10-epi-g-eudesmol 1625 – – – – – – – – – 0.1 A1-epi-cubenol 1631 – – – 0.1 – – 0.2 – – – Ag–eudesmol 1632 – – – – – – – 0.1 – – Apogostol 1653 – – – – – – – – 1.1 – A6,6-dimethyl-6(3-methylphenyl)-heptan-3-one 1642 – – – – 0.3 – – – – – Aepi-g-cadinol 1644 – – – – – – 1.9 – – 0.2 Aa-muurolol 1646 – – – 1.6 – 2.0 0.3 – – 0.1 A3-butylphthalide 1646 – – tr – – – – – – – Aa-cadinol 1656 – – – – – 3.6 3.7 – – – Atrans-calaminen-10-ol 1668 – – – – – – – 0.3 – – Atetradecanol 1674 – – – – – – – 0.5 – – Aa-bisabolol 1687 – – – – – – – – – 0.5 Aeudesm-4(15),7-dien-1b-ol 1686 – – – – – – 0.5 – – – Asenkyunolide 1724 – tr tr – – – – – – – A,Bmint sulfide 1741 – – – tr – 0.4 tr – – 0.5 Abenzyl benzoate 1760 – – – – – – 0.2 – – tr Amethyl hexadecanoate 1922 – – – – – – – 0.5 – – Ahexadecanoic acid 1962 0.1 tr 0.1 – 0.9 – 3.9 1.5 – 0.6 Blinoleic acid 2132 – – – – – – – 0.6 – 0.4 Bosthole 2138 – – tr – 10.9 – – 0.2 – 3.0 A,Blinolenic acid 2140 – – tr – – – – 0.6 – –

% of oil identified w97 w99 w98 w92 w90 w90 w84 w91 w88 w97

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 thoseof authentic samples; C ¼ observed RI and mass spectra matched with those in Kollmannsberger et al., 1998.

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species group, the Black Swallowtail butterfly (Papilio polyxenes), has been thoroughly studied to determine the specificchemical constituents involved in female attraction. Feeny et al. (1989) and Baur et al. (1993) found that host-searchingbehavior and alighting on leaves increased in the presence of certain volatile components of carrot oil (Daucus carota), butthese compounds alone did not enhance oviposition rates. This is because upon alighting, female Papilionids drum leafsurfaces with foretarsi to “taste” for certain non-volatile chemicals before depositing eggs (Feeny et al., 1983, 1988). Elec-troantennogram (EAG) studies with P. polyxenes females showed that both non-polar and polar fractions of the carrot oilproduced 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 whensearching for host plants. However terpinolene and myrcene, two non-polar components, produced good EAG spikes. Theyfound that landing frequency was greater on artificial leaves soaked with the four polar compounds sabinene hydrate, (Z)-3-hexenyl 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, 4-terpineol and bornyl acetate were present in only six of the nine species, while sabinene hydrate and (Z)-3-hexenyl acetatewere entirely absent (Table 2). Our results for P. indra host plants revealed that the non-polar pinenes (a and b), and b-phellandreneþ limonene were consistently present and frequently in highest proportions across the nine host species (Table2). These compounds emit strong citrusy/piney aromas characteristic of these plants. Combined pinene composition rangedfrom 1.0% (C. hendersonii) to 49.1% (Aletes acaulis), and b-phellandrene þ limonene composition ranged from 2.7% (C. hen-dersonii) to 39.4% (Tauschia parhshii). Myrcene, p-cymene, the b-ocimenes, terpinene, g-terpinolene, and the polar compounda-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 theprevalent non-polar constituent of the oils, instead of the polar constituents. We suspect that Indra Swallowtail females areattracted to appropriate hosts by the aromatic qualities of one compound, or a few of these compounds acting in synergy. Thehost shift from the ancestral family Rutaceae to the Apiaceae was likely facilitated by the prominent citrus/piney aromasemitted 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 anisearoma of this species. Dethier (1941) found that methyl chavicol was the best attractant for P. polyxenes. However his studyinvolved caterpillars instead of adults. Based on Dethier’s results and the high percent composition of these two compounds,

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.

V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547544

one or both are potential attractants for Indra Swallowtails. Dethier inferred that for a given Apiaceae host the mostprominent compounds of the oil were likely used as key olfactory attractants, and to human olfactory senses thesecompounds represent the characteristic aroma of the plant. We theorize that this is true for P. indra. Eight of the nine P. indrahosts we studied have citrusy or pine-like aromas, the exception being C. hendersonii, and amongst the essential oilcomponents 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 pathwaysare expected to be similar as seen for C. hendersonii (Table 2, Fig. 1). Indra Swallowtails are known to use a particular hostspecies across the host’s geographical range (pers. obs., W.H. Whaley) and are likely using the same chemical attractantsproduced 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 oilcompositions of the nine hosts (Table 3 and Fig. 1). These non-host species do not emit the citrusy/piney aromas, or the anisearoma (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, andmethyl chavicol were important separators due to presence in all or most of the host plants, while being absence and/or inlow concentrations in the non-host plants (Table 3). Individual compounds or synergistic combination of these may act asfemale attractants. Certain compounds in the four non-host species, e.g., (Z)-3-hexenol (Table 3), may act as deterrents tooviposition. However non-use of a plant species by an insect, even though its larvae could feed successfully on it, may be dueto lack of appropriate olfactory attractants so that females never make physical contact with the plant. For example, lepi-dopterists 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 PCAloadings of essential oil components produced results contrary to what was expected since the Lomatium, Cymopterus and

Table 3Aletes acaulis (Aaca), Cymopterus hendersonii (Chen-1, and Chen-2), Cymopterus panamintensis (Cpan), Lomatium rigidum (Lrig), Lomatium scabrum (Lsca),Musineontenuifolium (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 listedcomponents are frequently discussed in the text.

Components Aaca Chen-1 Chen�2 Cpan Lsca Lrig Tpar Mten Scap Targ Ldisa Ldasb Lutrb Lnevc

(Z)-3-hexenol – – – – – – – – – – 18.5 1.8 0.5 3.9hexanol – – – – – – – – – – 1.0 – 0.2 0.6furfural – – – – – – – – – – 0.4 1.0 – 0.4benzaldehyde – – 0.04 – – – – – – – 0.3 – – 0.1o-guaiacol – – – – – – – – – – 0.1 – 0.6 –

2-phenylethyl alcohol – – – – – – – – – – 0.2 – 0.3 –

citronellol 0.3 2.7 3.5 1.6 0.4 0.5 0.5 2.4 – – – – – –

citronellal – 1.2 0.9 0.9 0.1 0.1 0.1 0.4 – – – – – –

a-pinene 10.8 0.3 0.6 0.9 1.4 6.9 6.4 0.6 1.0 0.5 – – 0.1 0.2b-pinene 38.3 0.7 1.2 0.9 4.6 3.5 8.8 0.1 0.04 2.4 – – 0.1 0.1b-phellandrene D limonene 29.0 2.7 4.7 19.8 26 28.6 39.4 25.5 11.3 6.4 – – 0.2 4.0methyl chavicol 0.2 29.9 42.3 0.2 – 0.8 0.1 – – – – – – –

myrcene 8.1 2.1 1.6 30.0 8.2 3.9 2.6 1.0 0.2 3.0 6.0 0.1 0.1 1.6(Z)-b-ocimene 1.5 0.2 0.2 4.9 11.8 0.2 1.3 0.3 17.9 13.1 0.2 0.3 – 0.8(E)-b-ocimene 0.5 3.1 2.9 4.5 4.0 0.6 1.5 0.4 4.3 5.7 1.0 0.1 0.2 5.1g-terpinene 0.3 1.8 0.5 0.04 0.3 0.1 1.2 0.3 6.4 0.2 – 0.4 0.3 3.3terpinolene 0.3 0.2 0.04 0.2 0.8 0.8 0.8 0.2 1.2 9.2 – – 0.1 1.7linalool 0.5 1.2 – 3.6 – 0.8 – 2.3 1.2 – 0.6 0.5 0.4 0.4a-terpineol 1.1 0.7 0.6 0.2 0.5 0.1 0.1 0.4 0.3 0.3 0.2 0.8 0.6 0.24-terpineol 0.5 0.8 0.6 0.3 – 0.2 0.1 0.4 – – 0.1 1.7 11.2 0.8bornyl acetate – – – 0.6 3.7 2.4 0.7 1.1 – 0.2 – 0.6 – –

(E)-anethole 0.04 11.8 21.5 – – – – – – – – – – –

% partial composition 91.4 59.4 81.14 68.64 61.8 49.5 63.6 35.4 43.84 41 28.3 7.3 14 23.2

a Bairamian et al., 2004.b Asuming et al., 2005.c Beauchamp et al., 2007.

V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547 545

Tauschia species were not closely clustered. However as expected the two C. hendersonii samples clustered nicely (Table 3 andFig. 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 inFig. 1. These two indra host species are phenotypically very similar and often confused when encountered in the field. Theirsimilarity in essential oil composition and morphology may indicate a closer relationship than is presently represented bytheir placement in separate genera. To infer true relationships from our data would be tenuous, because phylogenies forfamily Apiaceae and specifically for genera Cymopterus, Lomatium, Aletes, Tauschia and Musineum, are in great flux (Downieet al., 2002; Soltis and Novak, 1997). Because phylogenies for the Apiaceae using morphological and molecular data are not inagreement, it is difficult to make comparisons with those based on secondary metabolites. However, we tend to concur withthe 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 IndraSwallowtail host plants. This may account for the observation that in the southern deserts of California P. indra fordi femalesare occasionally attracted to the Rutaceae species Thamnosma montana (pers. obs., W.H. Whaley). The result is attractionwithno apparent oviposition, a likely result of contact chemical “tasting” during foretarsi drumming of leaves. In these desertmountains anothermachaon member, the Desert Black Swallowtail butterfly (P. polyxenes coloro) uses T. montana as its mainhost. Not unexpectedly, b-phellandrene and limonene comprise as much as 64% of the essential oils of this species (Tuckeret al., 2005) and its leaves and stems emit strong lemony aromas. In the same regions P. p. coloro occasionally oviposits onthe P. i. fordi host plant C. panamintensis (pers. obs., W.H. Whaley) and produces viable offspring. Interestingly, duringinfrequent 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 theseT. montana-fed indra caterpillars survive to produce viable, fertile adults. It appears that certain citrusy essential oilcomponents 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

V. Dev et al. / Biochemical Systematics and Ecology 38 (2010) 538–547546

plants, e.g., the eight members of the machaon species group, suggests a very recent host shift from the Rutaceae, perhaps asearly 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 thesearomatic compounds are possibly important constituents involved in the attraction. Since Rutaceae is the primary host familyand the probable ancestral host family of Papilio, these essential oil constituents provide an excellent roadmap for furtherstudy. Of the P. machaon species group members only P. polyxenes has been studied to determine the volatiles that attractfemales to their host plants. Whether the predominant non-polar essential oil components or the less common polarcomponents are key attractants is a matter to be resolved using laboratory experiments with live females and purifiedessential oil compounds. Better understanding of the chemically stimulated host plant search behavior of P. indra, a speciesholding considerable interest amongst butterfly enthusiasts, may shed light on the evolution of this behavior amongst theother 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. Theauthors wish to thank Larry Blakely, John Emmel, Kenneth Davenport, DaveWikle, Paul Rumpa and Grace Kostel for collectingsome of the plants. Thanks to Floyd Howell, Staff Emeritus of the Chemistry Department, California State PolytechnicUniversity, Pomona, for assistance in the maintenance of the instruments.

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