1. INTRODUCTION

In Korea, four species of Pieris Schrank in the Pieridae are known: P. rapae (Linnaeus 1758), P. canidia (Sparrman 1768), P. melete (Ménétriés 1857), and P. napi (Linnaeus 1758) (Joo et al. 2021). Among them, the taxonomic status of Pieris napi was questioned since this species is widespread from Europe to Asia and North America and the incosistency in wing pattern elements among populations. Eitschberger (1983) and Tuzov et al. (1997) distinguished the Far Eastern populations of P. napi, including those in Korea, from the European population based on morphology. There have been also different opinions over the taxonomic classification of the Korean populations of P. napi (Kim et al. 2001; Lee 2005): the Korean population to be the same as the European P. napi (Joo et al. 2021); and proposing it as a distinct species, P. dulcinea (Butler 1882) (Lee 2005). These two species, P. napi, and P. dulcinea, are very similar to each other by wing shape and patterns, requiring careful observation of the androconia shapes for accurate identification (Tadokoro et al. 2014). These two species also easily interbreed in laboratory experiments and produce fertile offspring (Kuroda 2008, 2010). Recently, Si et al. (2023) conducted morphological and genetic analyses of the East Asian Pieris napi-complex that comprises more than four species, P. napi, P. dulcinea, P. nesis, and P. melete, and provided a diagnosis to differentiate P. dulcinea from P. napi. This suggests that the Korean population should be reclassified as P. dulcinea.

On Jejudo Island three species of Pieris are known: P. rapae, P. melete, and P. napi (Lee 2005;Kim et al. 2021). P. rapae primarily inhabits in the lowlands, while P. melete is found in low elevation of mountainous regions. The populations of P. napi sensu Lee (2005) occurred in the grasslands and shrublands around the highlands on Jejudo Island at altitudes above 1,000 meters (Lee 2005;Kim et al. 2021). This highland population of P. napi sensu Lee (2005) was recognized as an independent subspecies, hanlaensis (Okano and Pak 1968), and showed distinct morphological differences from the mainland populations (Lee 1982;Joo et al. 2021). Since the mainland populations of P. napi were reclassified as P. dulcinea, the highland populations of P. napi on Jejudo Island was brought to question.

In Japan, there have been various opinions regarding applying scientific names to the species of the P. napi-complex based on localities across the country (Shirozu 2006;Oda 2016). Tadokoro (2019) divided this complex into three distinct taxa: Pieris nesis nesis Fruhstorfer, 1909 (Honshu), a subspecies of Pieris nesis (southern Hokkaido), and Pieris pseudonapi Verity, 1911 (eastern Hokkaido, Sakhalin, southern Kurils).

Since there have been other species of Pieris napicomplex, we sequenced mitochondrial COI of the highland population on Jejudo Island and found that this population is grouped with the Honshu population in Japan, P. nesis. Consequently, we examined the genetic and morphological differences in the Pieris-napi complex between the mainland and Jejudo Island populations in Korea and report the occurrence of P. nesis in Korea.

2. MATERIALS AND METHODS

We examined 17 specimens preserved in the Department of Environmental Education at Mokpo National University, South Korea, along with specimens collected by the first author. Observations included external morphology and both male and female genitalia.

To compare the genetic differences with related species, genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). PCR was conducted using a T100TM Thermal Cycler (Bio- Rad, USA), with primers LCO1490 and HCO2198 (Folmer et al. 1994), and GainBlueTM HOT Start Pro Premix, 2x (GainBio, Daejeon, Korea) to amplify the partial COI gene (658 bp) of mitochondrial DNA. The PCR procedure followed the method described by Choi et al. (2021). The PCR products were purified using ExoSAP-IT^TM PCR Product Cleanup Reagent (Applied Biosystems, USA) and subsequently sent to Bioneer Co., Ltd. (Daejeon, Korea) for sequencing. The sequences were edited and aligned using MEGA 11 (Tamura et al. 2021). The COI sequences were identified in NCBI (National Center for Biotechnology Information) and used to calculate genetic differences between the species and their relatives within the genus.

Genetic distance and Maximum Likelihood (ML) analyses were conducted using 26 COI sequences. Sequences for 2 individuals were obtained from the BOLD Systems database (www.barcodinglife.org, Barcode of Life Data System). The remaining sequences were retrieved from the NCBI GenBank database (https:// www.ncbi.nlm.nih.gov/). Among the 26 sequences, 25 belong to species of the genus Pieris, while one sequence was used as an outgroup, Anthocharis scolymus (Butler 1866) within Pierinae. The sequence alignment was performed using the Muscle algorithm (Edgar 2004) in MEGA 11 (Tamura et al. 2021) with default settings. The genetic distance was estimated using MEGA 11 with the Kimura 2-parameter model (Kimura 1980). The ML analysis was constructed using IQtree 1.6.2 (Nguyen et al. 2015). A model test was initially conducted using IQ-tree to determine the bestfit model for the data. The ML analysis was then performed with 2,000 bootstrap replications to assess the robustness of the inferred tree. The voucher/specimen ID and GenBank accession numbers/sequence ID for the COI barcodes used in the analysis can be found in Table 1. The terminology of wing and genitalia follows that of Shirozu (2006). Abbreviations are as follows: TL, type locality; TS, type species; JJ, Jejudo Island.

3. TAXONOMIC ACCOUNTS

Order Lepidoptera Linnaeus, 1758

Family Pieridae Swainson, 1820

Genus PierisSchrank, 1801

PierisSchrank, 1801: 152, 161. TS: Papilio brassicae Linnaeus

Pieris nesisFruhstorfer, 1909 한라줄흰나비 (신칭) (Figs. 1, 2)

Pieris napi nesisFruhstorfer, 1909: 88; TL: Sapporo, Japan.

Pieris napi hanlaensisOkano and Pak, 1968: 65; Joo and Kim, 2002: 82; Joo et al., 2021: 307. TL: Mt. Halla- san, Korea.

Artogeia napi hanlaensis: Lee, 1982: 13.

Material examined. [JJ] 1♂, Seoquipo-si, Hallasan-ro (alt. 1,100 m), 22.V.2000; 1♂, Seoquipo-si, Hallasan-ro (alt. 1,100 m), 15.VII.2015; 1♂ 1♀, Seoquipo-si, Hallasan- ro near service area (alt. 1,100 m), 10.VII.2015; 10♂ Seoquipo-si, Hallsanro near service area (alt. 1,109 m), 3.VIII.2024; 1♂, Jeju-si, 1100 (alt. 1,509 m), 3.VIII.2024; 1♀, Seoquipo-si, Yeongsilro, near Busstop, 16.VII.1999; 1♀, Witseo-rum (alt. 1,700 m), 25.VII.2017.

Diagnosis. This species closely resembles P. dulcinea but is slightly smaller, with more prominent apical blackish spots on the upperside of the forewing that are not separated along the veins. Black scales are visible in the discoidal cell on the underside of the forewing, a feature rarely seen in P. dulcinea. Additionally, the black discal spots in spaces 1b and 3 on the underside of the forewing are more pronounced. Black scales are present along the veins on the underside of the wings, setting it apart from P. dulcinea.

Description. Wing expanse about 45 mm in spring brood and about 50 mm in summer brood. Spring form is relatively smaller than summer form. Labial palpus with an elongated third segment. Both wings white; forewing underside pale yellow (more whitish in summer), females show a stronger yellow hue; discoidal cell with black scales, denser toward the base.

Spring brood: Males (Fig. 1A, B). Apical blackish spot faint, separated from each vein; spot in space 3 nearly absent; outer spot very faint on the upperside. Hindwing outer margin slightly powdered black along the vein tips. Females (Fig. 1E, F): Apical spot darker, more pronounced, and thicker than in males, barely separated from the veins. Brownish-black markings on all veins more distinct than in males, more pronounced on the underside than the upperside, narrowing toward the edges.

Summer brood: Males (Fig. 1C, D). Apical blackish spot dark, extending to the outer margin and merging; spots in spaces 1b and 3 strongly developed, more pronounced on the underside than the upperside. Brownish- black scales along veins on the underside slightly developed. Outer spots absent. Females (Fig. 1G, H). Apical blackish spots heavily developed, dark brownish, and merged. Black spots in spaces 1b and 3 very strongly developed on both the upperside and underside. Brownish powder on all veins narrower on the upperside, slightly visible on the underside.

Male genitalia (Fig. 2A, B). Tegumen broad, with a distinct concave at almost its median part, distal margin moderately-sclerotized as a pair of sub-lobe shapedconvex; uncus basal width as wide as the distal part of tegumen, gradually tapering to a slender point.

Female genitalia (Fig. 3A, B). Posterior apophysis long and slender, extending beyond the 8th tergum; inner distal part of sterigma short, lobe-shaped, extending towards the center; inner basal part of sterigma with a rounded plate protrusion, connected at the basal margin; signum cordiform, with a broad and short terminal part, tapering moderately towards the base; P. dulcinea (Fig. 3C, D) differs by a narrower central area.

DNA phylogeny. DNA barcoding of the Pieris napicomplex revealed a genetic distance of 1.20% from P. dulcinea and 6.15% from P. melete. The intraspecific variances for P. napi and P. dulcinea were 0.13% and 0.18%, respectively (Table 1, Fig. 4). The phylogenetic tree indicated that the Pieris napi-complex is a monophyletic group, with P. napi being the sister species to P. dulcinea.

Ecology. The Pieris nesis populations on Jejudo Island emerge twice a year, in May-June and July-August, similar to their occurrence in the high mountains of mainland Korea. On Jejudo Island, the host plants are Arabis serrata Franch. & Sav. var. hallaisanensis (Nakai) Ohwi and Barbarea orthoceras Ledeb., which have been transplanted from the lowlands (Kim et al. 2021). Remarks. The morphological comparison of three populations in Japan, including forewing length, wing patterns, and scent gland structures, was summarized in Tatokoro (2019). Among these, the populations from Honshu (Japan) and two populations from Korea, mainland and highland on Jejudo Island showed that both Jejudo Island and Honshu (Japan) shared the characters such as more developed and fused apical blackish spots along veins on the forewing and a few markings on the discal cell of forewing underside while the Korean mainland populations showed relatively faint and separated apical blackish spots along veins on the forewing and sparse markings on the discal cell of forewing underside. These morphological differences suggest that the morphological similarity between Jejudo Island and Honshu is greater than in mainland Korea.

The highland butterfly species of Jejudo Island are mostly seen on the northern part of Korean Peninsula, predominantly belonging to the Palearctic region, specifically the Manchurian subregion. Many of these species are relict from the glacial period and these include more than 10 butterfly species (e.g., Plebejus argus (Linnaeus 1758), Argynnis niobe (Linnaeus 1758), Melanargia epimede Staudinger, 1892, Coenonympha hero Linnaeus, 1761, Lopinga achine (Scopoli 1763), Hipparchia autonoe (Esper 1784), Aphantopus hyperantus (Linnaeus 1758), Oeneis urda (Eversmann 1847), Hesperia comma (Linnaeus 1758)) (Joo and Kim 2002;Kim et al. 2013). Additionally, some species (e.g. a saturniid moth, Saturnia jonasii Butler, 1877) are occurring in the Japanese archipelago. This suggests that Jejudo Island had a complex geological history with the mainland and Japan, and it is likely that the White Butterfly (P. nesis) also might colonize Jejudo Island when it was connected to the Japanese archipelago.