Obligate pollination mutualisms with seed-parasitic insects are rare but provide classic systems for studying coevolution. In the fig–fig wasp association (Moraceae–Agaonidae) and the yucca–yucca moth association (Agavaceae–Prodoxidae), insect pollinators actively transfer pollen during oviposition, and their larvae consume a portion of developing seeds, leaving enough to ensure host reproduction (Janzen, 1979;Pellmyr, 1999).

A third obligate pollination–seed predation system was first documented by Kato et al. (2003): the association between Glochidion (Phyllanthaceae) and Epicephala (Gracillariidae). In this system, female moths actively collect pollen with their proboscis, deposit it onto the stigmas of female flowers, and subsequently oviposit through the ovary wall. The larvae consume only part of the seeds, allowing host plants to reproduce. Subsequent studies revealed that such interactions are not confined to Glochidion but also occur in other Phyllanthaceae lineages, including Phyllanthus (subg. Gomphidium) and Breynia, highlighting the broader significance of Epicephala –Phyllanthaceae obligate pollination mutualisms (Kawakita and Kato, 2004a;2004b;2006;2016).

In Korea, two Epicephala species have previously been reported: E. relictellaKuznetzov, 1979 and E. nudilinguaKawakita and Kato, 2016 (Kawahara et al., 2010;Lee and Jeun, 2022), but neither is known to act as a pollinator. We report the first discovery of an Epicephala pollinator associated with the Korean endemic species Glochidion chodoense C.S. Lee and H.T. Im (Fig. 1). Kim et al. (2016) categorized G. chodoense, restricted to Jindo-gun, Jeollanam-do, as Critically Endangered (CR). Despite its conservation status, the pollination biology of this species has remained completely unknown. We identified its pollinator as E. obovatellaKawakita and Kato, 2016, previously known only from Japan and Taiwan, based on morphological, behavioral, and molecular evidence.

Materials and Methods

Specimens were examined under a Leica Z16 APO stereomicroscope (Leica Microsystems, Wetzlar, Germany). Focusstacked image sets (≥50 frames per specimen) were captured with a Dhyana 400DC camera (Tucsen Photonics, Fuzhou, China). Image stacks were combined using Helicon Focus v8.2.18 (Helicon Soft Ltd., Kharkiv, Ukraine). Minor adjustments to brightness, contrast, and background were performed in Adobe Photoshop 26.0.0 (Adobe Inc., San Jose, CA, USA). Morphological terminology follows Kawakita and Kato (2016). All specimens examined are deposited in the Korea National Arboretum (KNA), Pocheon, Korea.

Total genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. For adult specimens, legs were used as the DNA source to minimize damage to voucher specimens. For eggs dissected from host plant fruits, whole eggs were homogenized for DNA extraction. The mitochondrial COI barcode region (658 bp) was amplified using the standard primer pair LepF1 and LepR1 (Hebert et al. 2004). PCRs were performed with Platinum Taq (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Amplicons were purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and sequenced by Macrogen Inc. (Seoul, Korea).

Sequences were assembled and aligned in Geneious Prime v2025.2.2 (Biomatters, Auckland, New Zealand). Pairwise genetic distances were calculated under the Kimura 2-parameter (K2P) model in MEGA 12 (Kumar et al. 2024). A maximum likelihood (ML) analysis was conducted in MEGA 12 using the General Time Reversible + Gamma + Invariant sites (GTR+G+I) model, selected with the built-in model selection tool, with 1,000 bootstrap replications to assess nodal support. The analysis included Korean E. obovatella sequences together with all Epicephala COI barcode sequences reported by Kawakita and Kato (2006, 2016) and deposited in Gen- Bank. Conopomorpha flueggella Li, 2011, Stomphastis labyrinthica (Meyrick, 1918), Melanocercops ficuvorellaYazaki, 1926, and Cuphodes diospyrosella (Issiki, 1957) were used as outgroups, following Kawakita and Kato (2016).

Taxonomic Accounts

Family Gracillariidae Stainton, 1854

Subfamily Ornixolinae Kuznetzov and Baryshnikova, 2001

Genus EpicephalaMeyrick, 1880

EpicephalaMeyrick, 1880: 137, 168.

Type species: Epicephala colymbetellaMeyrick, 1880.

IrainaDiakonoff, 1955.

LeiocephalaKuznetzov and Baryshnikova, 2001.

Epicephala obovatellaKawakita and Kato, 2016 (Fig. 2, 3) 조도만두가는나방(신칭)

Epicephala obovatellaKawakita and Kato, 2016: 101 (type locality – Wakayama Prefecture, Japan; holotype ♀, KYO; Paratypes – same data as holotype 1♂, 1♀).

Diagnosis. Wingspan 7.0–10.0 mm. Head ground color brown, densely covered with white scales dorsally. Female proboscis with abundant trichoid sensilla. Thorax white dorsally. Forewing brown with narrow white band. Apical margin reddish brown with central black dot and short white streak near dorsum. Hindwing brown. Wing margin with grayish brown cilia.

Male genitalia. Tegumen elongate, elliptical, about three times as long as wide. Cucullus rounded rectangular, densely covered with hairs. Sacculus oval, about 0.7× length of cucullus, with a distal finger-like projection bearing a row of spines. Vinculum U-shaped; saccus elongate-oval, acute at apex. Aedeagus slender and straight without cornutus.

Female genitalia. Lamella postvaginalis forming two reniform lobes, each about half the length of the seventh abdominal segment. Seventh abdominal segment truncated cone-shaped. Corpus bursae oval, with a triangular signum medially located. Apophyses posteriores about 1.48× length of apophyses anteriores. Ovipositor long and slender, laterally toothed, apex angular.

Material examined. 6♀, 1♂, San 162-1, Namdong-ri, Imhoemyeon, Jindo-gun, Jeollanam-do, South Korea, N34°22'3.78" E126°09'55.06", 22.VI.2023, G.Y. Han et al.; 1♀, ditto, 10.VI. 2024, G.Y. Han et al.; 3♀, ditto, 11.VI.2024, G.Y. Han et al.; 3 ♀, ditto, 12.VI.2024, G.Y. Han et al.; 5♀, ditto, 18.VI.2024, S.S. Euo et al.; 3♀, ditto, 19.VI.2024, S.S. Euo et al.; 2♀, ditto, 25.VI.2024, S.S. Euo et al.; 1♀, ditto, 22.VII.2024, S.S. Euo et al.

Distribution. Japan, South Korea, Taiwan.

DNA barcode. Sequences were uploaded to NCBI (Gen- Bank accession numbers: PX241574–PX241576).

Remarks. The Korean specimens agree well with the original description by Kawakita and Kato (2016). Both male and female genitalia were nearly identical to those of Japanese specimens, with only minor variation in female genital length ratios. DNA barcode analysis further placed the Korean specimens within the intraspecific variation of E. obovatella and clearly separated from other congeners. Based on both morphological and molecular evidence, the Korean population is therefore identified as E. obovatella.

Results

As previously reported for other Epicephala–Glochidion systems (Kato et al. 2003;Kawakita and Kato, 2004a, 2004b, 2006, 2016), only females were observed to perform active pollination in E. obovatella associated with G. chodoense. Females were observed on female flowers exclusively between 19:00 and 22:00, transferring pollen to stigmas with the proboscis (Fig. 3A) and ovipositing through the ovary wall (Fig. 3B). Pollen grains were observed attached to the proboscis of females (Fig. 2D) but not in males (Fig. 2C).

Pairwise K2P genetic distances between Korean samples and all available populations of E. obovatella ranged from 0.0 to 3.15% (mean 1.44%). These values fall within the reported intraspecific variation of E. obovatella (≤4.12%) and are clearly distinct from interspecific distances (≥4.3%) with other Epicephala species (Kawakita and Kato, 2016).

Maximum likelihood analysis recovered E. obovatella encompassing both previously recognized subclades as a wellsupported monophyletic group (Fig. 4). The overall topology was consistent with the two major subclades of E. obovatella previously reported by Kawakita and Kato (2006, 2016). Korean specimens from Jindo Island clustered within Subclade 1b (bootstrap support: 67%), forming a compact group with short branch lengths, and were closely related to Japanese and Taiwanese populations.

Discussion

Our study provides the first evidence of pollination by Epicephala in Korea and demonstrates that Glochidion chodoense participates in the well-known Glochidion–Epicephala obligate mutualism. Field observations of female moths performing proboscis-mediated pollen deposition and oviposition confirmed active pollination behavior, establishing the species as a specialized pollinator.

This discovery documents E. obovatella as new to Korea, representing an extension of its known distribution. Importantly, this record shows that E. obovatella, previously known to utilize G. obovatum and G. rubrum in Japan and Taiwan, also occurs on G. chodoense. This finding highlights that pollinator ranges can span multiple host species and do not necessarily coincide with the distribution boundaries of a single Glochidion species. From a conservation perspective, the confirmation of obligate pollination in G. chodoense emphasizes that both the plant and its pollinator must be considered in management strategies for this species.

Previous studies recognized two major subclades within E. obovatella (Subclade 1 and Subclade 2; Kawakita and Kato 2006, 2016). Our analysis shows that Korean E. obovatella are included within Subclade 1. Subclade 1 is further divided into two lineages (Subclade 1a and 1b), and the Korean specimens grouped with other members of Subclade 1b. Patterns of host plant use showed that Subclade 2 was exclusively associated with G. rubrum, while Subclade 1a was restricted to G. obovatum. In contrast, Subclade 1b contained populations linked to multiple Glochidion species. This finding reinforces previous reports that host use in E. obovatella is not strictly consistent across its range (Kawakita and Kato, 2006;2016) and further substantiates the mosaic pattern of host association within a single nominal species.