Philippine Journal of Science 150 (S1): 111-119, Special Issue on Biodiversity ISSN 0031 - 7683 Date Received: 07 Sep 2020 Cytochrome C Oxidase Subunit I and II (COI and COII) Sequences Support Recognition of Papilio chikae Igarashi and Papilio hermeli Nuyda as Distinct Species John Vincent B. Cabusas1, Aimee-Lynn A. Barrion-Dupo2,3, Romnick A. Latina1, and Barbara L. Caoili1* 1Institute of Weed Science, Entomology, and Plant Pathology College of Agriculture and Food Science 2Environmental Biology Division, Institute of Biological Sciences College of Arts and Sciences 3Entomology Section, Museum of Natural History University of the Philippines Los Baños College, Laguna 4031 Philippines Papilio chikae and P. hermeli are two endemic swallowtail butterflies in the Philippines found primarily in the Cordillera region and Mindoro Island, respectively. Their species status is still subject to debate due to opposing interpretations of their genitalia and morphological traits. This study aims to delineate these taxa through the use of mitochondrial cytochrome C oxidase subunit I (COI) gene marker, with additional information on their cytochrome C oxidase subunit II (COII) gene. The COI nucleotide sequences from our Philippine P. chikae samples were surprisingly identical to P. hermeli EF514465.1 on the GenBank, whereas our P. hermeli samples showed a 100% similarity with P. hermeli EF514464.1. Based on our phylogenetic analysis, P. chikae and P. hermeli exhibit distinct COI barcodes supported by a high bootstrap value (85%). The P. hermeli voucher specimen with GenBank Acc. No. EF514465.1 clustered with our P. chikae samples, separate from the cluster containing our P. hermeli specimens. Nonetheless, the 3.1–3.4% genetic distance observed in the COI region supported by the formation of two separate clusters suggests species status for these taxa. Moreover, species-specific molecular markers obtained from the COII region displayed a 2.4% distance between these two taxa. These molecular markers could therefore be utilized to distinguish P. chikae from P. hermeli. Nevertheless, a thorough examination of their morphology, behavior, and ecology is still imperative to firmly establish their taxonomic status. Keywords: divergence rate, DNA barcodes, Luzon peacock swallowtail butterfly, species delineation INTRODUCTION account of their high species richness and endemism, well-documented taxonomy, and a high degree of host- Unraveling diversification patterns and biogeographical plant specialization (Condamine et al. 2012). Currently, history has been partly made possible by studying ideal subgenus Achillides is comprised of up to 25 species and candidates, such as species of Papilio (Achillides) at least a hundred subspecies distributed across Asia, butterflies, also known as “peacock swallowtails,” on particularly the Indo-Australian archipelago. *Corresponding Author: [email protected] 111 Philippine Journal of Science Cabusas et al.: DNA Barcodes of Distinct Luzon Vol. 150 No. S1, Special Issue on Biodiversity Peacock Swallowtails In the Philippines, endemism has been documented on and Treadaway (2004) as a standard nomenclature two Papilio species, commonly referred to as Luzon reference of Philippine Papilionidae, CITES (Convention peacock swallowtails. P. chikae Igarashi was first on International Trade in Endangered Species of Wild discovered in 1965 in Mount Santo Tomas, and later near Fauna and Flora) also proposed to amend the current the peaks of Benguet, Mountain Province, and Southern listing of P. chikae to A. chikae chikae (CITES 2018). The Cordillera (Page and Treadaway 2004). P. hermeli – absence of papiliochrome wing pigments in Achillides which was first described by Nuyda in 1992 – has been (present in other subgenera of Papilio) was used as a observed to inhabit Mount Halcon and Mount Baco, basis for the amendments, in addition to its distinct larval situated on the northern and southern parts of Mindoro and pupal forms (Igarashi and Fukuda 2000; Page and Island, respectively (Nuyda 1992; Treadaway 1995; Treadaway 2004). Bauer and Frankenbach 1998; Page and Treadaway 2004; Treadaway and Schröder 2012). Both peacock Delineation of species using mitochondrial genes has swallowtails are present at montane forests at high been preferred due to fewer intron interferences, few altitudes where ferns, lichens, orchids, and mosses thrive recombinations, and its haploid transmission (Hebert (Page and Treadaway 2003). They are speculated to feed et al. 2003). The COI gene is nowadays commonly on Euodia spp., which belong to the Family Rutaceae used to identify lepidopteran species known for low (Igarashi 1984; Page and Treadaway 2004). sequence divergence (Hebert et al. 2003; Hajibabaei et al. 2006; Litman et al. 2018). Combined molecular and Papilio chikae and P. hermeli share many similar traits. morphological analyses of lepidopterans have been shown Golden-green scales are evenly scattered throughout to increase the rate of species identification than using their black body and wings on the dorsal side. Ventrally, either approach singularly (Litman et al. 2018). the dark-brown forewing has pronounced white bands that taper towards the inner margins, with dark veins The two Luzon peacock swallowtails have become spreading across the wing. The hindwing holds a series of alarmingly vulnerable to various threats from collectors, red, wavy lunules on the edges, mixed with bluish-green traders, researchers, and environmentally destructive patches along the sub-margin. The tail elongates towards practices by locals (Tsukada and Nishiyama 1982; the end, narrowing at its base. The underside of their Treadaway 1995; CITES 2018). P. chikae has been hindwing is black, where a portion is spotted by whitish included in the International Union for Conservation of scales marked by pale purple spots enclosed on red-orange Nature (IUCN) Red Data Book of threatened species, lunules along the margins (Collins and Morris 1985). The CITES list of endangered species, and Appendix A of female has more red spots on the dorsal hindwing than European Union regulation on trading with species the male, but the male has larger bluish-green patches. of wild fauna and flora. Recent proposals have also The two swallowtails, however, are differentiated based been made to include P. hermeli in the CITES list of on the distinctive whitish-gray bands on the ventral side endangered species (CITES 2018). Hence, this study of the forewing of P. hermeli, which in P. chikae is less was designed to provide baseline information relevant apparent, and tapers to pale blue in the inner margin of to the conservation efforts and strategies of these the forewing (CITES 2018). swallowtails. In the context of these two Luzon peacock swallowtails, their molecular data could be used in Disagreement over the taxonomic ranking of these inferring phylogenies through the use of analytical two taxa has persisted for decades. Some authors methods such as the maximum likelihood (ML), which treated the differences in genitalia across sexes of provides an array of sophisticated evolutionary models these two swallowtails as significant, thus classifying fit for understanding sequence evolution of organisms them as separate species (Treadaway 1995; Bauer (Yang and Rannala 2012). While mitochondrial COII and Frankenbach 1998; Häuser et al. 2005). Others sequences were unavailable for both P. chikae and asserted the conspecificity of these two taxa, noting a P. hermeli prior to this study, COI sequences were close resemblance in their appearance, granting them available on GenBank for P. hermeli, but none for separation at subspecies level (Page and Treadaway P. chikae (Fontanilla et al. 2014). The objectives of 2004; CITES 2018). The subspecies concept was this study were: 1) to compare the Luzon peacock created to characterize geographical variations based swallowtails specimens based on partial mitochondrial on morphology but objective metrics to discriminate COI and COII nucleotide sequences; 2) to compare subspecies are lacking, failing to show unique derived nucleotide and amino acid sequences; and 3) to evolutionary units (Braby et al. 2012). In 2004, Page construct a phylogenetic tree based on these molecular and Treadaway elevated Achillides from subgenus to operational taxonomic units. genus level, changing the nomenclature of Papilio hermeli to Achillides chikae hermeli. Referring to Page 112 Philippine Journal of Science Cabusas et al.: DNA Barcodes of Distinct Luzon Vol. 150 No. S1, Special Issue on Biodiversity Peacock Swallowtails MATERIALS AND METHODS model and illustrated via ML using MEGA7 software version 7.0.26 with 1,000 bootstrap replications (Kumar et al. 2016). Source of Test Insects Voucher specimens of two individuals each of Papilio chikae (UPLBMNH LEP-04639 and UPLBMNH LEP- 04640) and P. hermeli (UPLBMNH LEP-04641 and LEP-04642) were provided by the Museum of Natural RESULTS History, University of the Philippines Los Baños (UPLB), The partial COI and COII gene fragments with molecular College, Laguna. sizes of approximately 700–750 base pairs (bp) and 800 bp, respectively were amplified. Upon trimming, Molecular Identification of the Swallowtail Species a 658-nucleotide long partial COI gene sequence and Molecular analyses were conducted at the Insect 676-nucleotide-long partial COII gene were generated Pathology and Molecular Biology Laboratory, Institute for all the specimens. of Weed Science, Entomology, and Plant Pathology, College of Agriculture and Food