Taxonomic Review of the Genus Caloptilia Hübner, 1825 (Lepidoptera: Gracillariidae) with Descriptions of Three New Species and Seven Newly Recorded Species from Korea

Simple Summary Caloptilia is a genus of the family Gracillariidae, one of the most diverse groups of Microlepidoptera, with more than 2000 described species worldwide. The species of this family are known as leafminers because they mine into the leaves of various host plants. Caloptilia has been poorly studied in Korea, with 50% fewer known species than those in the neighboring countries, viz., China, Russia, and Japan. In this study, we aimed to clarify fauna of the genus Caloptilia in Korea by describing three new species and seven newly recorded species in the country. In addition, we list all known species from Korea, along with images of the adult specimens and genitalia. Abstract In this study, 29 species of Caloptilia Hübner, 1825, belonging to the family Gracillariidae, were recognized in Korea. Among these, three species, i.e., C. purpureus sp. nov., C. koreana sp. nov., and C. xanthos sp. nov., are described as new to science. In addition, seven species of this genus are reported for the first time in Korea. All known species were enumerated, based on their available information. Adult specimens and genitalia of the new and newly recorded species were examined and described using all available information.


Introduction
The family Gracillariidae is one of the most diverse groups of Microlepidoptera, with more than 2000 described species belonging to 112 genera worldwide [1]. The genus Caloptilia is the second largest group in the family, comprising more than 450 species worldwide [1]. They are cosmopolitan in distribution, with 180 species, ca. 40% of the total known species, found in the Palearctic region. They can be distinguished from the other genera based on the following observations (i) adults in a resting posture raising their head upward; (ii) abdomens (A) with a membranous tergite and sclerotized sternite on A8; (iii) females with a pair of signa; (iv) final instar larvae with leaf rolling behavior that pupate within the leaves of host plants [2,3].
Among them, the genus Caloptilia was not well studied until Shin et al. [5] enumerated 19 species from Korea, with four newly reported species. Of them, two species, C. fidella and C. illicii, were misidentified as Gracillaria albicapitata and C. sapporella, respectively. Recently, Lee and Jeun (2022) [16] reported two newly recorded species: C. pyrrhaspis (Meyrick) and C. syrphetias (Meyrick). To date, 21 species of this genus have been reported in this country.
In this study, a more intense investigation of the genus Caloptilia was conducted, using the results of an examination of the big collection from the 1970s. We found three new species, C. purpureus sp. nov., C. koreana sp. nov., and C. xanthos sp. nov., and recorded seven species for the first time in Korea. These include C. acericola , C. celtidis Kumata, 1982 [2], C. dentata Liu and Yuan, 1990 [20], C. kadsurae Kumata, 1966 [18], C. monticola Kumata, 1966 [18], C. recitata   [21], and C. soyella (van Deventer, 1904) [22]. Here, we provide a description of these three new species along with the taxonomic arrangements and annotated checklist of all known species. In addition, information on collection locality, host plant, and distributional range is provided.

Materials and Methods
The specimens examined in this study were deposited at the Systematic Entomology Laboratory of Hannam University, Daejeon, Korea (HNSUEL). Male and female genitalia were dissected and mounted with Euparal solution, following the procedure described by Holloway et al. [23]. Images of the adults were taken using a digital camera (Canon EOS 600D, Canon Inc., Ota, Tokyo, Japan), and images of the genitalia were captured using a digital camera attached to a microscope, LEICA M205C (Leica Microsystems, Wetzlar, Hesse, Germany) and refined using Photoshop CS5 software. Terminology for the genitalia of both sexes was followed after Landry [24]. Diagnosis. This species is similar to C. theivora in appearance, but can be distinguished by the costal margin with narrow yellowish streak after the blotch apically and metallic purple ground color of forewing. In the genitalic structures, the new species is different from the latter by having numerous long setae on apex of valva in male genitalia and two signa on corpus bursae in female genitalia.

Description of New Species
Adult (Figure 1a). Forewing length 3.5 mm. Head grayish ochreous and tinged with violet reflection; frons pale yellowish white and face yellow; maxillary palpus yellow with a tiny fuscous spot on the median part; labial palpus yellow with apex fuscous; antenna pale ochreous and scape fuscous. Thorax pale grayish fuscous; fore legs fuscous with whitish tarsus; hind femur yellow with a fuscous median broad band; hind tibia and tarsus pale ochreous.
Forewing ground color grayish fuscous with violet reflection; a triangular goldish yellow blotch, covering nearly half of forewing, extends to wing fold with blackish edges and 2-3 blackish dots on apical margin; a goldish yellow streak begins just after blotch apically, occupying 1/4 of forewing and broader at median without any edges; veins with Sc and R1 close, R3 and R4 very close, M2 stalked with M3. Hindwing is similar to C. koreana.
Male genitalia (Figure 2a). Tegumen ovate with parallel side to apex and as long as vinculum or slightly longer than vinculum. Valva up-curved, elongated, rectangular shape with parallel side from base to 1/3 of valva, dilated on apex, dorsal margin more elongated on apex and apical margin straight; long and slender setae along apical margin to a half of ventral margin, more sparse ventrally, more short and strong setae sparse along the apical margin. Vinculum triangular shape with acute apex. Juxta small and 'u' shaped. Aedeagus bar-shaped, apex somewhat rectangular shape, slightly sclerotized and slightly bent inward without any cornuti.
Female genitalia (Figure 3a). Papillae anales moderated, long setae on vertex sparser with a pair of long scales on each, laterally of base; posterior apophyses triangular shaped broadening to base and as long as anterior. Ostium bursae small in opening size; sternum VIII divided into two lateral parts and shot as long as 1/6 of posterior apophyses. Dustus bursae very slender, as long as 2.5 times of corpus bursae and coiled near corpus bursae. Corpus bursae ovate with two signa on median; signa with different size each other, blunt and rather rectangular apex.
Insects 2022, 13,1107 3 of 26 Male genitalia (Figure 2a). Tegumen ovate with parallel side to apex and as long as vinculum or slightly longer than vinculum. Valva up-curved, elongated, rectangular shape with parallel side from base to 1/3 of valva, dilated on apex, dorsal margin more elongated on apex and apical margin straight; long and slender setae along apical margin to a half of ventral margin, more sparse ventrally, more short and strong setae sparse along the apical margin. Vinculum triangular shape with acute apex. Juxta small and 'u' shaped. Aedeagus bar-shaped, apex somewhat rectangular shape, slightly sclerotized and slightly bent inward without any cornuti.
Female genitalia (Figure 3a). Papillae anales moderated, long setae on vertex sparser with a pair of long scales on each, laterally of base; posterior apophyses triangular shaped broadening to base and as long as anterior. Ostium bursae small in opening size; sternum VIII divided into two lateral parts and shot as long as 1/6 of posterior apophyses. Dustus bursae very slender, as long as 2.5 times of corpus bursae and coiled near corpus bursae. Corpus bursae ovate with two signa on median; signa with different size each other, blunt and rather rectangular apex.
Remarks. This species was reared from Sageretia theezans (L.) Brongn. of the family Rhamnaceae in this study.
The triangular yellow costal blotch of the new species almost the same as C. theivora; however, costal margin with narrow yellowish streak after the blotch apically. In the new species, long setae on apical of valva dense along margin, whereas C. theivora naked at median of apical margin. In female genitalia, there are two signa on corpus bursae in new species, while C. theivora has only a signum. Etymology. The specific name is derived from the color of forewing, purple. Distribution. Korea (endemic, Yeosu-si). Host plants. Sageretia theezans (L.) Brongn. [Rhamnaceae] in Korea (in this study). Remarks. This species was reared from Sageretia theezans (L.) Brongn. of the family Rhamnaceae in this study.
The triangular yellow costal blotch of the new species almost the same as C. theivora; however, costal margin with narrow yellowish streak after the blotch apically. In the new species, long setae on apical of valva dense along margin, whereas C. theivora naked at median of apical margin. In female genitalia, there are two signa on corpus bursae in new species, while C. theivora has only a signum.  1♀, Lake Yuklim, Gwangneung, 7 September 2017 (leg. Lim, Kim, Lee, Shin, Roh), gen. slide no. HNUSEL-5575-coll. KNAE.
Diagnosis. This species is similar to C. chrysolampra, but can be distinguished by the more extended size of the yellow costal blotch of the forewing and female genitalia with well sclerotized ductus bursae in the new species, whereas they are very slender and membranous in the latter.
Adult (Figure 1b). Forewing length 3 mm in female. Head grayish ochreous and occiput gray with flat and large scales; frons white and face pale yellowish white; maxillary palpus white with a tiny fuscous spot on the first segment laterally; labial palpus silvery white with a tiny fuscous spot laterally and fuscous apically; each segment of antenna grayish fuscous, pale ochreous basally. Thorax grayish with a pale ochreous median line; tegular more darkened to fuscous posteriorly; fore and middle legs fuscous with whitish tarsus; hind legs pale ochreous with tiny fuscous spots apically.  Forewing ground color grayish fuscous, tinged with violet reflection; a yellow dorsobasal streak from base to 1/6 of forewing; large and rounded yellow blotch begins at 1/3 to base and extends to wing fold, the interval between blotch and dorsal margin broadens more apically and tiny blackish dots sparsely on costal margin; venation with R 1 arising from before middle, R 2 arising from 2/3 of cell, R 3 , R 4 and R 5 not close, M2 and M3 stalked, M3 and CuA close near discal end of cell. Hindwing lanceolate and ground color silvery dark gray; vein with Rs reaching to subapex of costa, M1 and M2 form a branch, M3 distant from M2, then close to Cu.
Insects 2022, 13,1107  Diagnosis. This species is similar to C. chrysolampra, but can be distinguished by the more extended size of the yellow costal blotch of the forewing and female genitalia with well sclerotized ductus bursae in the new species, whereas they are very slender and membranous in the latter.
Adult ( Figure 1b). Forewing length 3 mm in female. Head grayish ochreous and occiput gray with flat and large scales; frons white and face pale yellowish white; maxillary palpus white with a tiny fuscous spot on the first segment laterally; labial palpus silvery white with a tiny fuscous spot laterally and fuscous apically; each segment of antenna grayish fuscous, pale ochreous basally. Thorax grayish with a pale ochreous median line; tegular more darkened to fuscous posteriorly; fore and middle legs fuscous with whitish tarsus; hind legs pale ochreous with tiny fuscous spots apically.
Forewing ground color grayish fuscous, tinged with violet reflection; a yellow dorsobasal streak from base to 1/6 of forewing; large and rounded yellow blotch begins at 1/3 to base and extends to wing fold, the interval between blotch and dorsal margin broadens more apically and tiny blackish dots sparsely on costal margin; venation with R1 arising from before middle, R2 arising from 2/3 of cell, R3, R4 and R5 not close, M2 and M3 stalked, M3 and CuA close near discal end of cell. Hindwing lanceolate and ground color silvery dark gray; vein with Rs reaching to subapex of costa, M1 and M2 form a branch, M3 distant from M2, then close to Cu.
Male genitalia. Unknown. Female genitalia (Figure 3b). Papillae anales somewhat acute on vertex, short and sclerotized on base; posterior apophyses broaden at base and slightly sclerotized; anterior apophyses as long as posteriores. Lamella postvaginalis with a well sclerotized flap, the flap reversed pot shaped; ostium bursae with same width as ductus bursae. Male genitalia. Unknown. Female genitalia (Figure 3b). Papillae anales somewhat acute on vertex, short and sclerotized on base; posterior apophyses broaden at base and slightly sclerotized; anterior apophyses as long as posteriores. Lamella postvaginalis with a well sclerotized flap, the flap reversed pot shaped; ostium bursae with same width as ductus bursae.
Ductus bursae strongly sclerotized with many horizontal wrinkles, as long as corpus bursae, slightly curved to small 'S' shape and broadened to near the corpus bursae. Corpus bursae ellipse form and slightly sclerotized near ductus bursae with two signa on 1/4 of corpus bausae; signa falciform, somewhat straight, and acute apically.
Etymology. The specific name is derived from the type locality, Korea. Distribution. Korea (endemic, Gwangneung). Host plants. Unknown. Remarks. This species is similar to C. chrysolampra, but can be distinguished by the size of yellow costal blotch of the forewing. The yellow costal blotch of new species extends more to nearly apex, and forewing ground color grayish fuscous. In female genitalia, ductus bursae well sclerotized with a pair of falciform shaped two signa. Diagnosis. This species is similar to C. theivora, but can be distinguished by rather long corpus bursae with two signa in female genitalia Adult (Figure 1c). Forewing length 4 mm. Head covered with pale grayish fuscous. Thorax grayish fuscous with two ochreous lateral line on tegular; legs fuscous and white; middle tarsus white with a fuscous median band. Forewing purplish fuscous with a yellow costal blotch; a yellow costal blotch begins at 1/3 to base and is triangular with very acute apex without edges; venation with R 1 arising from 1/3 of cell, R 3 , R 4 and R 5 not close, M1 distant from M2+M3 at base, M2 and M3 stalked, M3 and CuA not close. Hindwing lanceolate and ground color silvery dark gray; veins with Rs reaching to apex of costa, M1 and M2 form a branch, M3 distant from M2.
Male genitalia. Unknown. Female genitalia (Figure 3c). Papillae anales slightly swollen with rounded process on vertex; posterior apophyses as long as anterior and slightly broadened at base. Ostium bursae on a sclerotized ellipse form disc and small in opening size; antrum slightly sclerotized and reduced. Ductus bursae as long as 1.7 times of corpus bursae and slender. Corpus bursae elongated, very long and slender with a lot of minute spinules on whole surface; signa dislocated from each other, well sclerotized and base more elongated to inner side.
Etymology. The specific name is derived from the yellowish patch of forewing. Distribution. Korea (endemic, Muan-gun). Host plants. Unknown. Remarks. This species is similar to C. theivora, but the new species has two signa on corpus bursae of female genitalia, whereas the latter has a signum. This species is similar to the members of the genus Caloptilia in appearance. In the new species, the yellow triangular costal blotch in the forewing is almost the same as C. theivora; however, it can be distinguished by the small yellow spots along the costal margin from the end of blotch to near the subapex, then tinged with yellowish scales toward apex. In female genitalia, there are two singa on corpus bursae, instead of only one sigum in the latter.  Gin-no-lang-ga-neun-na-bang Caloptilia acericola Kumata, 1966: 2-3 [19]. TL: Hokkaido, Japan. TD: EIHU (Holotype; Paratypes).
Remarks. This species was reared from Celtis sinensis Persoon of the family Cannabaceaee in this study.
Remarks. This species was reared from K. japonica Dunal of the family Magnoliaceae in this study.
Autumnal: Forewing ground color purplish brown; basal-costa margin dark brown; a boundary line of yellow blotch more obscure than aestival form; a row of tiny blackish spots along costa margin, a spot on 2/3 to base, which is more distinct and larger.
Male genitalia (Figure 2f). Tegumen concave to inner surface, as long as 2/3 of vinculum and apex round. Valva moderate in shape with other species of this group; spatulate shaped, base narrow and broadens to apex with slender scales densely. Vinculum elongated, triangular and apical round. Aedeagus slender, bar-shaped and narrowed to apex; a long and slender cornuti on apical part, three of short and stout cornuti near base and the last one more elongated with bifurcated apical part.
Remarks. This species was collected from Ailanthus altissima (Mill.) [Simaroubaceae] with pupal cocoon on the back side.
Remarks. This species was reared from Lespedeza cyrtobotrya Miq. of the family Fabaceae in this study.
Remarks. This species was reared from Celtis sinensis Persoon of the family Cannabaceae in this study.
Forewing length

Discussion
The genus Caloptilia has not been studied extensively in Korea to date, when compared with that in the neighboring countries, especially regarding the number of known species, e.g., Japan has more than 50 reported species [1]. The first record of Caloptilia in Korea, which included five species, was reported in 1983 by Park. Later, Park and Han (1986) reported six species of Gracillaridae. In the 2000s and the 2010s, Park and Lee (2001) [70] and Sohn (2007) [44] reported one and four species, respectively. Shin et al. (2015) [5] enumerated 19 species of the genus, with four newly recorded species from Korea. All the known species from Korea including this study, were enumerated with synonymies, distribution and host plant respectively (File S1).
The genus Caloptilia can be distinguished from the other genera in the family Gracillariidae, based on their males having weakly membranous 7th and 8th abdominal segments, and females with signa present in the corpus bursae of their genitalia. Among the species of Caloptilia, the identification keys can be used to identify each species; however, slight differences exist. In addition, most species of Caloptilia morphologically resemble each other, with triangular patches on the forewing, a pair of signa in the female genitalia, and valva dilated toward the apex in the male genitalia. The host plant might be an exact identification key for Gracillariidae moths, because they have high host plant specificity (Davis, 1987 [116]; Brito et al., 2016 [117]), except for some polyphagous species. In this study, we reared a total of nine species of Caloptilia from host plants when they were in larval stages. Among them, three species that were investigated provided new host plant information, and one of them, Ailanthus altissima (Mill.) was recorded as a new host plant of the family Gracillariidae. Based on the host plant survey in this study, all the known host plants were summarized for the gracillariid species (Table S1). In total 106 species of 15 families were investigated for host plants of Caloptilia in the world. Most of them consisted of Salicaceae (27 species), Sapindaceae (23 species), Ericaceae (14 species), Betulaceae (12 species) and Fagaceae (11 species) [1]. The monophagous character is an obvious characteristic in the genus Caloptilia, but some species were shown to be polyphagous in this study. Caloptilia stigmatella were investigated with three families of host plants, and both C. recitata and C. magnoliae had two families, respectively.
In future research in Korea, DNA barcodes will function as exact identification keys for microlepidoptera. In this study, we tried to extract the DNA barcodes targeting all species of Caloptilia in Korea. However, it was difficult to get successful results, especially on some old specimens or other specimens, due to unknown reasons. We are now preparing and collecting more fresh material. After this, we will try to study the taxonomies and conduct systematic study with species delimitation and DNA data in future.