Essential oil composition of nine Apiaceae species from western United States that attract the female Indra Swallowtail butterfly (Papilio indra)

Biochemical Systematics and Ecology 38 (2010) 538–547

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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: [email protected] (W.H. Whaley). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.05.010

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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.

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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

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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 – – – – – – – – – – – – – –

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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)

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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,

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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

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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

Chen2

Cpan

Lsca

Lrig

Tpar

Mten

Scap

Targ

Ldisa

Ldasb

Lutrb

Lnevc

(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

– – – – – – 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

a b c

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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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. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, fourth ed. Allured Publishing Corporation, Carol Stream, Illinois. Asuming, W.A., Beauchamp, P.S., Descalzo, J.T., Dev, B.C., Dev, V., Frost, S., Ma, C.W., 2005. Essential oil composition of four Lomatium Raf. species and their chemotaxonomy. Biochem. Syst. Ecol. 33, 17–26. Bairamian, S., Beauchamp, P.S., Descaizo, J.T., Dev, B.C., Dev, V., Frost, C.F., Nguyen, C.V., 2004. California Lomatiums Part III. Composition of the hydrodistilled oils from two varieties of Lomatium dissectum. Isolation of a new hydrocarbon. J. Essent. Oil Res. 16, 461–468. Baur, R., Feeny, P., Stadker, E., 1993. Oviposition stimulants for the black swallowtail butterfly: identification of electrophysiologically active compounds in carrot volatiles. J. Chem. Ecol. 19 (5), 919–937. Beauchamp, P.S., Dev, B.C., Dev, V., Midland, S.L., Sims, J.J., 2007. California Lomatiums, Part VII. Analysis of the essential oils of Lomatium nevadense (Watson) J. Coulter et Rose var. parishii (J. Coulter et Rose) Jepson. Isolation of trans-dauc-8-en-11-ol, a new sesquiterpene alcohol and naturally occurring 20 ,30 ,30 trimethyl-20 ,30 dihydroangelicin. J. Essent. Oil Res. 19, 117–124. Beauchamp, P.S., Chea, E., DimaanoDimaano, J.G., Dev, V., Ly, B., Miranda, A.E., Whaley, W.H., 2009. Essential oil composition of six Lomatium species attracted to indra swallowtail butterfly (Papilio indra): principal component analysis against essential oil composition of Lomatium dissectum var. multifidum. J. Essent. Oil Res. 21, 535–542. Berenbaum, M.E., 1995. Chemistry and oligophacy in the Papilionidae. In: Scriber, J.M., Tsubaki, Y., Lederhouse, R.C. (Eds.), Swallowtail Butterflies: Their Ecology and Evolutionary Biology. Scientific Publishers, Gainesville, Florida, pp. 27–38. Cronquist, A., Holmgren, N.H., Holmgren, P.K., 1997. Intermountain Flora, vol. 3A, New York Botanical Garden, Bronx, NY. 446 pp. De Pasquale, F., Siragusa, M., Abbate, L., Tusa, N., De Pasquale, C., Alonzo, G., 2006. Characterization of five sour orange clones through molecular markers and leaf essential oils analysis. Scient. Hort 109, 54–59. Dethier, G., 1941. Chemical factors determining choice of food plants by Papilio larvae. Am. Nat. 75, 61–75. Dev, V., Ly, B., Miranda, A.E., Whaley, W.H., 2007. Lomatium grayi and Indra Swallowtail Butterfly. Composition of the essential oils of three varieties of Lomatium grayi (J.M. Coult et Rose) J.M. Coult et Rose. J. Essent. Oil Res. 19, 244–248. Downie, S.R., Hartman, R.L., Son, F., Katz-Downie, D.S., 2002. Polyphyly of the spring-parsleys (Cymopterus): molecular and morphological evidence suggest complex relationships among the perennial endemic genera of western North American Apiaceae. Can. J. Bot. 80, 1295–1324. Emmel, J.F., Shields, O., 1978. Larval food plant records of Papilio zelicaon in western United States, and further evidence of the conspecificity of P. zelicaon and P. gothica. J. Res. Lepid 17, 56–67. Feeny, P., Rosenberry, L., Carter, M., 1983. Chemical Aspects of Oviposition Behavior in Butterflies. In: Ahmad, S. (Ed.), Herbivorous Insects. Academic Press, New York, pp. 27–76. Feeny, P., Sachdev, K., Rosenberry, L., Carter, M., 1988. Luteolin 7-0-(600 -0-malonyl)-b-D-glucoside and trans-chlorogenic acid: oviposition stimulants for the black swallowtail butterfly. Phytochemistry 27, 3439–3448. Feeny, P., Stadler, E., Ahman, I., Carter, M., 1989. Effects of plant odor on oviposition by the black swallowtail butterfly, Papilio polyxenes (Lepidoptera: Papilionidae). J. Insect Behav. 2, 803–827. Hansson, B.S., 1995. Olfaction in Lepidoptera. Experientia 51, 1003–1027. Hansson, B.S., 2002. A bug’s smell – research into insect olfaction. Trends in Neurosciences 25 (5), 270–274. Heinz, C.A., 2008. Host plant odor extracts with strong effects on oviposition behavior in Papilio polyxenes. Entomol. Exp. Appl. 128, 265–273. Hickman, J.C. (Ed.), 1993. The Jepson Manual: Higher Plants of California. University of California Press, Berkeley, CA, 1400 pp. Hitchcock, C.L., Cronquist, A., 1973. Flora of the Pacific Northwest. University of Washington Press, Seattle, WA, 730 pp. Honda, K., 1995. Chemical basis of differential oviposition of lepidopterous insects. Arch. Insect Biochem. Physiol. 30, 1–23. Jermy, T., 1984. Evolution of insect/host plant relationships. Am. Nat. 124, 609–630. Kearney, T.H., Peebles, R.H., Howell, J.T., McClintock, E., 1960. Arizona Flora, second ed. University of California Press, Berkeley, CA, 1085 pp. Kirbaslar, G., Kirbaslar, S., 2004. Composition of Turkish bitter orange and lemon leaf oils. J. Essent. Oil Res. 16, 105–108. Kollmannsberger, H., Lorenz, M., Weinreich, B., Nitz, S., 1998. The flavour composition of Manzanilla de la Montana (Helenium aromaticum) from Chile. Adv. Food Sci. 90, 122–131. Lota, M., de Rocca Serra, D., Tomi, F., Casanova, J., 2001. Chemical variability of peel and leaf essential oils of 15 species of mandarins. Biochem. Syst. Ecol. 29, 77–104. Papaj, D.R., 1986. Conditioning of leaf-shape discrimination by chemical cues in the butterfly, Battus philenor. Anim. Behav. 34, 1281–1288. Rausher, M.D., 1978. Search image for leaf shape in a butterfly. Science 200, 1071–1073.

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

547

Ruberto, G., Biondi, D., Piattelli, M., Rapisarda, P., Starrantino, A., 1994. Essential oil of the new citrus hybrid Citrus clementina x C. limon. J. Essent. Oil Res. 6, 1–8. Ruberto, G., Renda, A., Piattelli, M., Rapisarda, P., Starrantino, A., 1997. Essential oil of two new pigmented citrus hybrids, Citrus clementina x Citrus sinensis. J. Agric. Food Chem. 45, 467–471. Saxena, K.N., Goyal, S., 1978. Host-plant relations of the citrus butterfly Papilio demoleus L.: orientational and ovipositional responses. Entomol. Exp. Appl. 24, 1–10. Scriber, J.M., 1973. Latitudinal gradients in larval feeding specialization of the world Papilionidae (Lepidoptera). Psyche 80, 355–373. Shapiro, A.M., Masuda, K.K., 1980. The opportunistic origin of a new citrus pest. Calif. Agric. 34 (6), 4–5. Soltis, P.S., Novak, J., 1997. Polyphyly of the Tuberous Lomatiums (Apiaceae): cpDNA evidence for morphological Convergence. Syst. Bot. 22 (1), 99–112. Sperling, F.A.H., 1987. Evolution of the Papilio machaon species group in western Canada. Quaest. Entomol. 23, 198–315. Sperling, F.A.H., Feeny, P., 1995. Umbellifer and composite feeding in Papilio: phylogenetic frameworks and constraints on caterpillars. In: Scriber, J.M., Tsubaki, Y., Lederhouse, R.C. (Eds.), Swallowtail Butterflies: Their Ecology and Evolutionary Biology. Scientific Publishers, Gainesville, Florida, pp. 299– 306. Tucker, A.O., Maciarello, M.J., Landrum, L.R., 2005. The essential oil of Mojave Desert Rue, Thamnosma montana Torr.et Frem. (Rutaceae), from Arizona. J. Essent. Oil Res. 17, 366–367. 2005. Verzera, A., Alessandra, T., Zappal, M., Condurso, C., Cotroneo, A., 2005. Essential oil composition of Citrus meyerii Y. Tan. and Citrus medica L. cv. Diamante and their lemon hybrids. J. Agric. Food Chem. 53 (12), 3890–4894. Whaley, W.H., 2000. An illustrated guide to geographical variations in the Indra Swallowtail butterfly and its larval host plants. Annu. Meet. Pac. Slope Sect. Lepidopterists Soc.. http://research.uvu.edu/whaley/ June 3.