Unpalatability of Delias Butterflies (Lepidoptera: Pieridae) in the Highlands of New Guinea: Between-Species Variation in Unpalatability

Home , Cepora, Cepora laeta, Delias, Delias belladonna, Delias hyparete, Delias splendida

Biogeography 21. 22–26. Sep. 20, 2019

Unpalatability of Delias butterflies (Lepidoptera: Pieridae) in the highlands of New Guinea: between-species variation in unpalatability

Sadaharu Morinaka1*, Erniwati2, Nobuhiro Minaka3, and Tadashi Miyata4

1Saitama Study Center, The Open University of Japan, Saitama, Kawaguchi, 333-0805, Japan 2Entomology Laboratory, Zoology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), West Java, 12710, Indonesia 3Institute for Agro-Environmental Sciences, NARO, Ibaraki, 305-8517, Japan 4Graduate School of Bioagricultural Sciences, Nagoya University, Aichi, 464-8601, Japan

Abstract: Various butterfly species that have warning colors on their wings have been suggested to be unpalatable/toxic to predators; however, this unpalatability has only been confirmed experimentally in a few species. The present study examined the unpalatability of Delias species from the highlands of New Guinea using caged avian predators Pycnonotus aurigaster and Leiothrix lutea. The birds ate 9-19 butterflies of six Delias species from New Guinea, and ate palatable Mycalesis horsfieldii butterflies much more frequently thanDelias butterflies, but these predators ate only 1-3 butterflies ofDelias species from Bali. The results suggested that Delias species are unpalatable to predators but that this unpalatability is moderate among species from the highlands of New Guinea compared with those from Bali. We considered that well-defined mimics of Delias species are less common in the highlands of New Guinea, because of their moderate unpalatability.

Key words: Delias, feeding assay, mimicry, New Guinea, Pieridae, unpalatability.

Butterflies of the genus Delias Hübner, 1819 (Lepidoptera: species in Oriental and Australian regions. Pieridae), which are distributed from South India to the islands We focused on Delias species native to the highlands of of the South Pacific including the mainland of New Guinea New Guinea, where they showed high species abundance and the Australian subcontinent, have diversified markedly (Parsons, 1998; Braby & Pierce, 2007) and tendencies to (Talbot, 1928-1937; Yata, 1981; Parsons, 1998; Braby & gather near swamps or river sides (Morinaka,1993; Parsons, Pierce, 2007; Müller, et al., 2012). The genus includes over 1998). Here, they showed various wing markings that were 200 species classified into 22 species groups by Yagishita et similar or different between the east to west highlands of New al. (1993) on the basis of an earlier systematic study based Guinea (Morinaka,1993; Morinaka, et al., 2001; Morinaka, et on morphological characteristics (Talbot, 1928-1937), and al., 2011). The aim of this study was to determine the degree 24 species groups based on molecular phylogeny (Braby & of unpalatability of Delias species from the highlands of New Pierce, 2007). Molecular phylogeny generally supported their Guinea. To this end, we carried out feeding assays with these current classification (Morinaka et al., 2002; Braby & Pierce, species using wild birds and compared their unpalatability 2007; Müller et al., 2012; Morinaka et al., 2017). with that of Delias species from Bali. Delias generally have white wings with a black margin on Materials and Methods the upper-sides, which is common to pierids, being contrasted with bright, variegated markings in red, yellow, and/or other Feeding assays were carried out very similarly to those de- colors on the undersides as characteristics of the genus (Talbot, scribed by Morinaka et al. (2018). 1928-1937). Various well-defined mimics of Delias butter- Palatability assays flies have been identified, suggesting their unpalatability and Palatability was examined in a series three of assays. aposematism of their wing markings (Dixey, 1920; Talbot, Assay A: Dried specimens of Delias microsticha Rothchild 1928-1937; Yata, 1981; Yen, 2005). & Jordan, 1904 from the highlands of the Arfak mountains We demonstrated marked unpalatability in Delias species located in the west part of New Guinea; Delias oraia Doherty, distributed on Bali and Timor, and that they formed mimicry 1891, Delias sambawana Rothschild, 1894, and Mycalesis rings (Morinaka et al., 2018). However, the marked unpal- horsfieldii Moore, (1892) from Bali were also used. The spe- atability of Delias species may not be common to all Delias cies D. oraia and D. sambawana were used for comparison −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− with D. microsticha, and M. horsfieldii belonging to Satyrinae *Corresponding author: [email protected] (Nymphalidae), which were used as palatable species. Indi-

– 22 – Sadaharu Morinaka, Erniwati, Nobuhiro Minaka and Tadashi Miyata viduals of D. microsticha were collected from the highlands of from the Seg River in the highlands located in the central part the Arfak mountains by local catchers, and transported in fold- of New Guinea and D. oraia from Bali were used. Individuals ed paper at room temperature for less than a week to Bali. The of D. oraia were used for comparison with D. toxopei, with species D. oraia and D. sambawana were collected from Bali the latter collected by local catchers, and transported in folded and stored at room temperature for a few weeks. Sooty-head- paper at room temperature for less than a week to Japan. D. ed bulbul, Pycnonotus aurigaster obtained from Bali was used oraia were collected on Bali and stored at room temperature as a predator. for a few weeks. Red-billed leiothrix, Leiothrix lutea, pur- Assay B: Fresh specimens of D. microsticha, Delias liga- chased from a pet shop in Tokyo, Japan, was used as predators ta Jordan, 1904, Delias kenricki Talbot, 1937, Delias pratti in the assays carried out in 1997 (i.e., before the Invasive Al- Kenrick, 1909, and Delias castaneus Kenrick, 1909 from the ien Species Act became effective in 2005). highlands of the Arfak mountains and M. horsfieldii from Bali Delias species from the highlands of New Guinea used in were used. Delias species collected from the Arfak mountains the assays are shown in Fig. 1. were kept alive in a cooled container and transported to Bali Training birds over a few days. Individuals of M. horsfieldii from Bali were P. aurigaster used in assays A and B were obtained from kept alive in folded paper at room temperature for 1-2 days. P. Bali. Bulbuls generally consume various foods including in- aurigaster obtained on Bali were used as predators. sects in the field; therefore, they are considered to be suitable Assay C: Dried specimens of Delias toxopei Roepke, 1955 for feeding assays. Assays A and B were conducted in a shed

Fig. 1. Delias species in the highlands of New Guinea used in these assays. A: Delias microsticha, B: Delias ligata, C: Delias castaneus, D: Delias pratti , E: Delias kenricki, F: Delias toxopei.

– 23 – Unpalatability of Delias butterflies in the highlands of New Guinea on Bali. The birds were kept individually in cages made of Results wood and bamboo for approximately 2 months in order to acclimatize them to the captive environment. Every bird was fed In palatability assay A, the birds learned to reject dried bod- commercially available mash meal for birds ad libitum, a piece ies of D. microsticha after eating 9 or 13 bodies. Birds learned of banana every 2 days, and two crickets once a week. Fresh to reject dried bodies of Delias from Bali after eating 1-3 bod- water was provided ad libitum and replaced every evening. In ies. Single birds ate 28-37 bodies of M. horsfieldii, possibly the 2 weeks before palatability assays, each bird was trained up to satiation (Table 1). The results using Delias species and every day to eat food that was stabbed by the tip of a piece of M. horsfieldii from Bali were almost the same as the results of bamboo. Specifically, a live cricket was skewered onto the tip previous feeding experiments (Morinaka et al., 2018). Signif- of a bamboo stick and held near the beak of a bird, and then icant differences were observed in the number of butterflies the bird was allowed to eat it. Crickets were given to birds eaten among D. microsticha, Delias species from Bali and M. repeatedly until they ceased eating (Morinaka et al., 2018). horsfieldii, but not between Delias species from Bali (Table 2). The red-billed leiothrix, L. lutea used in assay C is insectiv- In assay B, the birds learned to reject the fresh bodies of orous and one of the easier wild birds to keep in a cage. The each species such as D. microsticha, D. ligata, D. kenricki, D. birds were kept individually in cages made of wire and plastic pratti, and D. castaneus after comsuming 9-15 bodies. On the for 1 month or more in order to acclimatize them. Every bird other hand, single birds ate 30-38 fresh bodies of M. horsfield- was fed commercially available mash meal and water for birds ii (Table 3). The number of butterflies eaten was significantly ad libitum. different between each of these Delias species and M. horsfiel- Palatability assay dii, but not among each Delias species (Table 4). In the palatability assays, the birds were not fed until 14:00, at which time the assays were started. The birds used in the Table 1. Assay A: Number of dried butterflies eaten by P. aurigaster. present study may have learned that some Delias butterflies Different individuals of P. aurigaster were used in the different trials. are distasteful in the wild before being captured. Therefore, Species (locality) Trial No. of butterflies eaten D. microsticha in order to eliminate the possible effects of wing markings on 1 13 males (Arfak mountains*) palatability assays, the wings of butterflies were removed with 2 13 males scissors just before the assay. The butterfly body was skew- 3 9 males ered onto the tip of a bamboo piece and presented to a bird 4 13 males (Morinaka et al., 2018), as performed in the training phase in D. oraia (Bali) 1 3 males assays A and B. A butterfly body was placed into a feedbox 2 2 males in assay C. When the bird pecked and swallowed at least part 3 1 male of the butterfly body, we recorded that the sample was eaten. 4 1 male Feeding was repeated for each butterfly species until the bird D. sambawana (Bali) 1 3 males 2 1 male did not eat an additional butterfly body within 5 minutes. The 3 1 male total number of butterflies of each species eaten by a single 4 2 males bird was used in analyses. Different assays used different Mycalesis horsfieldii (Bali) 1 37 individuals** individual birds; 2-4 birds were used per single species of 2 37 individuals** butterflies. Living birds were released back into the wild after 3 30 individuals** assays A and B. 4 28 individuals** Statistical analysis * The highlands of the Arfak mountains are located in the western part of New Guinea. Data were analyzed using R 3.2.1 (R Core Team, 2015). ** Sex not determined. For each of the assays, a generalized linear model was used in order to test for palatability differences among butterfly spe- Table 2. Pairwise comparisons of palatability among butterfly species in palatability assay A. cies. A Poisson distribution was used as the error distribution. Species 1 Species 2 Estimate (SE) z-value Significance The log function was used as the link function. The dependent D. microsticha M. horsfieldii -1.01 (0.169) -6.002 *** variable used in the models was the number of butterflies of D. oraia M. horsfieldii -2.94 (0.388) -7.572 *** the species eaten by a single bird. The butterfly species was D. sambawana M. horsfieldii -2.94 (0.388) -7.572 *** used as the nominal independent variable. P-values were cor- D. oraia D. microsticha -1.93 (0.405) -4.759 *** rected for multiple comparisons by Tukey’s method using the D. sambawana D. microsticha -1.93 (0.405) -4.759 *** R package ‘multcomp’ (Hothorn et al., 2008). D. sambawana D. oraia <0.001 (0.535) 0.000 n. s. ***P < 0.001. n. s., not significant; SE, standard error.

– 24 – Sadaharu Morinaka, Erniwati, Nobuhiro Minaka and Tadashi Miyata

In assay C, individual birds learned to reject dried butterfly 1999). bodies of D. toxopei after eating 12 or 19 bodies. On the other In a broad area including Bali and Timor where Delias hand, birds learned to reject dried bodies of D. oraia from Bali species with marked unpalatability are found (Morinaka et al., after eating 1-3 bodies (Table 5). The number of butterflies 2018), various lepidopteran species that very closely resemble eaten was significantly different forD. toxopei (Table 6). the sympatric Delias species are known to exist. For example, Prioneris autothisbe Hübner, (1826) resembles D. oraia DISCUSSION from Bali (Morinaka & Yata, 1994), and Cepora laeta Hew- To date, Delias species have been considered unpalatable itson, (1862) resembles Delias splendida Rothschild, 1894, and that their bright wing markings are aposematic (Dixey, Delias eileenae Joicey & Talbot, 1926 (Dixey, 1920; Talbot, 1920; Talbot, 1928-1937; Yata, 1981; Parsons, 1998). Howev- 1928-1937; Yata, 1981), and Delias lemoulti Talbot, 1931 er, it was unknown whether various Delias species are similar- (Yata, 1981) from Timor. Cepora temena Hewitson, (1861) ly unpalatable. resembles D. oraia and D. sambawana from the Lesser Sunda We found that Delias species from the highlands of New Islands (Dixey, 1920; Talbot, 1928-1937) between Bali and Guinea were unpalatable compared with M. horsfieldii, but Timor. Prioneris sita C & R Felder, (1865) resembles Delias they were only moderately unpalatable/toxic compared with eucharis Drury, (1773) in South India (Dixey, 1920; Talbot, Delias species from Bali. The present results demonstrated 1928-1937), and Prioneris thestylis Doubleday, (1842) resem- that the degree of unpalatability varied among Delias species. bles Delias belladonna Fabricius, (1793) found from North Differences in unpalatability among species of the same ge- India to China and Burma (Dixey, 1920; Talbot, 1928-1937). nus in Lepidoptera have been reported, for example, among These species are viewed as Batesian mimics. species of the genus Pieris (Kingsolver, 1987; Lyytinen et al., In contrast, in the highlands of New Guinea, very few

Table 3. Assay B: Number of fresh butterflies eaten by P. aurigaster. Table 4. Pairwise comparisons of palatability among butterfly species in Different individuals of P. aurigaster were used in the different trials. palatability assay B. Species (locality) Trial No. of butterflies eaten Species 1 Species 2 Estimate (SE) z-value Significance D. microsticha (Arfak mountains*) 1 12 males D. microsticha M. horsfieldii -0.95 (0.163) -5.794 *** D. ligata M. horsfieldii -1.18 (0.179) -6.636 *** 2 15 males D. kenricki M. horsfieldii -1.03 (0.168) -6.103 *** 3 12 males D. pratti M. horsfieldii -1.11 (0.174) -6.409 *** 4 13 males D. castaneus M. horsfieldii 1.26 (0.245) 5.141 *** D. ligata D. microsticha 0.24 (0.209) 1.138 n. s. D. ligata (Arfak mountains) 1 11 males D. kenricki D. microsticha 0.08 (0.200) 0.400 n. s. 2 10 males D. pratti D. microsticha -0.17 (0.205) -0.816 n. s. 3 11 males D. castaneus D. microsticha 0.314 (0.268) 1.170 n. s. D. kenricki D. ligata -0.16 (0.213) -0.741 n. s. 4 9 males D. pratti D. ligata 0.07 (0.217) 0.325 n. s. D. kenricki (Arfak mountains) 1 13 males D. castaneus D. ligata 0.0756 (0.278) 0.274 n. s. 2 12 males D. pratti D. kenricki -0.087 (0.209) -0.417 n. s. D. castaneus D. kenricki 0.234 (0.271) 0.862 n. s. 3 12 males D. castaneus D. pratti 0.147 (0.275) 0.534 n. s. 4 11 males ***P < 0.001. n. s., not significant; SE, standard error. D. pratti (Arfak mountains) 1 12 males Table 5. Assay C: Number of dried butterflies eaten by Leiothrix lutea. 2 11 males Different individual predators were used in the different trials. 3 10 males Sympatric Species of predator Locality Trial No. of butterflies eaten 4 11 males Species Leiothrix lutea Seg River* D. toxopei 1 12 males D. castaneus (Arfak mountains) 1 9 males 2 19 males 2 10 males Bali D. oraia 1 3 males Mycalesis horsfieldii (Bali) 1 30 individuals** 2 2 males 3 1 male 2 30 individuals** * Highlands located in the central part of New Guinea. 3 38 individuals** 4 36 individuals** Table 6. Pairwise comparisons of palatability among butterfly species in * The highlands of the Arfak mountains are located in the western part palatability assay C. of New Guinea. Species 1 Species 2 Estimate (SE) z-value Significance ** Sex not determined. D. toxopei D.oraia 2.05 (0.446) -4.591 *** ***P < 0.001.; SE, standard error.

– 25 – Unpalatability of Delias butterflies in the highlands of New Guinea lepidopteran species resembling Delias species are known, the western part of New Guinea Island. Biogeography. 13: although they have aposematic markings similar to those of 31-34. Delias species from Bali. We suggest that this is due to the Morinaka, S., Erniwati, Minaka, N., Miyata, T. 2018. moderate unpalatability of Delias species in the highlands. Mullerian mimetic radiation of Delias butterflies The differences in unpalatability among Delias species (Lepidoptera: Pieridae) in Bali and Timor. Entomol. Sci. 21: from Bali, Timor, and the highlands of New Guinea are possi- 396-405. bly due to phylogenetic constraints. The Delias species used Morinaka, S., Erniwati, Minaka, N., Miyata, T., Hoshizaki, S. in these assays are all endemic to the highlands of New Guin- 2017. Phylogeography of the Delias hyparete species group ea. They belong to four species groups that are phylogeneti- (Lepidoptera: Pieridae): complex historical dispersals into cally isolated from each other (Morinaka et al., 2002; Braby & and out of Wallacea. Biol. J. Linn. Soc. 121: 576-591. Pierce, 2007; Müller et al., 2012); almost all species of these Morinaka, S., Funahashi, K., Ginarsa I. K., Miyata, T., groups are endemic to the highlands of New Guinea. On the Tanaka, K. 2001. Delias butterflies (Lepidoptera, Pieridae) basis of the observed moderate unpalatability of multiple De- cohabiting in the highlands of New Guinea Island. Tyo Ga. lias species groups, the unpalatability of Delias species in the 52: 127-135. highlands of New Guinea might be the original characteristic Morinaka, S., Miyata, T., Tanaka, K. 2002. Molecular of the genus Delias. If this is not the case, it is possible that phylogeny of the Eichhorni group of Delias Hubner, 1819 evolution towards a decrease in unpalatability arose multiple (Lepidoptera: Pieridae). Mol. Phylogenet. Evol. 23: 276– times in the highlands of New Guinea. Moreover in queen 287. butterflies (Danaus gilippus Cramer, (1776)), variations in Morinaka, S., Yata, O. 1994. A New Subspecies of Prioneris unpalatability are known to be caused by differences in larval autothisbe (HÜBNER) (Lepidoptera, Pieridae) from Bali, host plants (Ritland, 1994). To determine the reason for these Indonesia. Jpn. J. Ent. 62: 22-28. differences, the unpalatability of Delias species including oth- Müller, C. J., Matos-Maraví, P. F., Beheregamy, L. B. 2012. er species groups should be further clarified. Delving into Delias Hübner: fine-scale biogeography, phylogenetics and systematics of the world’s largest Acknowledgments butterfly genus. J. Biogeogr. 40: 881-893. We thank Hari Sutrisno, Kenichiro Horio, Tadao Matsumoto, Parsons, M. J. 1998. The butterflies of Papua New Guinea: Isao Makibayashi, Osamu Yata, and Yukio Ishikawa for their en- their systematics and biology. Academic Press, London. couragement. Sugihiko Hoshizaki spent time holding discussions R Core Team. 2015. R: a language and environment for with S. M., and gave helpful comments on the draft. statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: http://www.R-project. References org/ Braby, M. B., Pierce, N. E. 2007. Systematics, biogeography Ritland D. B. 1994. Variation in Palatability of Queen and diversification of the Indo-Australian genus Delias Butterflies (Danaus Gilippus) and Implications Regarding Hübner (Lepidoptera: Pieridae): phylogenetic evidence Mimicry. Ecology. 75: 732-746. supports an ‘out-of-Australia’ origin. Syst. Entom., 32: 2-25. Talbot, G. 1928-1937. A monograph of the pierine genus Dixey, F. A. 1920. The geographical factor in mimicry. Trans. Delias. British Museum, London. R. Entom. Soc. Lond., 1920: 208-211. Yagishita, A, Nakano, S., Morita,, S. 1993. An illustrated list Hothorn, T., Bretz, F., Westfall, P. 2008 Simultaneous of the genus Delias Hübner of the world. Nishiyama, Y. (Ed.) inference in general parametric models. Biomed. J., 50: Khepera Publishers, Tokyo. Text 384 pp. Illustrated lists 346–363. 409 pp. (In Japanese.) Kingsolver, J. G. 1987. Predation, thermoregulation, and wing Yata, O. 1981. Pieridae. In: Tsukada, E. (Ed) Butterflies of the color in pierid butterflies.Oecologia 73: 301–306. South East Asian islands, Volume 2. pp. 206-438 for text, Lyytinen, A., Alatalo, R. V., Lindström, L., Mappes, J. 1999. plates 1-84. Plapac, Tokyo. (In Japanese.) Are European white butterflies aposematic? Evol. Ecol. 13: Yen,, S-H., Robinson, G. S., Quicke, D. L. J. 2005. The 709–719. phylogenetic relationships of Chalcosiinae (Lepidoptera, Morinaka, S. 1993. Observations on the “puddling behavior” Zygaenoidea, Zygaenidae). Zoo. J. Linn. Soc. 143: 161-341. of Delias species (Lepidoptera, Pieridae) in the central (Received May 14, 2019; Accepted June 18, 2019) highlands of Irian Jaya. Tyo Ga. 44: 89-96. Morinaka, S., Erniwati, Ginarsa, I. K. 2011. Co-occurring Delias butterflies (Lepidoptera: Pieridae) at Arfak Mts. in

– 26 –