First Report of the Immature Stages of the Leaf-Mining Genus Subclemensia Kozlov, 1987 (Lepidoptera: Incurvariidae), with a Re-Illustration of the Type Species and a Generic Concept Discussion Based on Immature Characters

Simple Summary Caterpillars and pupae are important sources of evidence for the identification of primitive moths, and they can often provide quite different but useful morphological characters from those of adults. Incurvariidae is one of the most primitive groups of extant Lepidoptera, but half of the genera lack any information on caterpillars and pupae. It is important to increase the knowledge on the immature stages of Incurvariidae. Subclemensia Kozlov, 1987 is one of the monotypic genera in Incurvariidae. In this study, the caterpillar and pupa of the type species of Subclemensia are reported for the first time. The leaf mine, host plant and its biological characteristics are also provided. DNA barcodes were generated to help with the species delimitation. The adult male and female genitalia are re-illustrated by color photography to supplement the original line drawings. Abstract The immature stages of primitive Lepidoptera can provide quite different but often useful morphological evidence and synapomorphies from those of adults. Incurvariidae is one of the most primitive lineages of extant Lepidoptera, which is species-poor but highly diverse, but half of the genera lack any information on immature stages. New knowledge on the immature stages of the family is expected to provide useful morphological evidence and synapomorphies to stabilize the generic nomenclature. Subclemensia Kozlov, 1987 is one of the monotypic genera in Incurvariidae. In this study, the immature stages of the type species of Subclemensia are reported for the first time. The leaf mine, host plant and its biological characteristics are also provided. DNA barcodes were generated to aid the species delimitation. The adult male and female genitalia are re-illustrated by color photography to supplement the original line drawings. The generic concepts of Subclemensia and other related genera are discussed based on immature characters.


Introduction
Incurvariidae is a species-poor but highly divergent family. The family has 54 species in 12 genera [1][2][3], including Plesiozela Karsholt & Kristensen, 2003 [4], which was recently moved into the family based on transcriptome phylogenetic analysis [5]. Most of the genera are represented by less than five species, or even by a single species. Immature stages of primitive Lepidoptera can provide quite different but often useful morphological evidence and synapomorphies from those of adults. However, little is known about the immature stages of half of the genera of Incurvariidae, such as Plesiozela, Protaephagus Scoble, 1980 [6], Perthida Common, 1969 [7], Simacauda Nielsen & Davis, 1981 [8], Basileura Nielsen & Davis, 1981 [8] and Subclemensia Kozlov, 1987 [9]. Therefore, it is of specific importance to discover

Materials and Methods
More than 20 leaf mines and early-instar larvae were collected on Betula platyphylla in Binggou Forest Farm (118.86 • E, 40.56 • N), Chengde, Hebei Province, China, in May 2020, to study the characteristics of the larvae and leaf mines. In early September, the larvae in portable cases descended the tree onto the ground to find a place for overwintering. These larvae were collected and placed in an outdoor net cage, and they emerged in mid-May of the following year. Immature larval specimens were kept for dissection and morphological study. Three first-instar larvae in cases and two mature larvae were examined. All the specimens were kept at Shandong Normal University (SDNU).
Genitalia and larvae preparations were made based on the method described by Li (2002) [16], using 10% NaOH solution to boil and digest internal tissues, and then transferring to water to clean up, stain with eosin and/or thiazole black and dehydrate with anhydrous ethanol. Then, the tissue was fixed with xylene and sealed with neutral gum to create permanent slides. Venation slides were created in a similar way but could be treated without 10% NaOH. Photos of the adult specimens were taken using a Canon EOS 5D Mark IV camera plus a Canon MP-E 65 mm lens (Canon Inc., Tokyo, Japan) and a Leica M165C stereo microscope (Leica Microsystems Ltd., Heerbrugg, Switzerland) to reveal the metallic luster under different light on the wings. Larvae, portable cases, pupae and early leaf mines were photographed with a Leica M165C stereo microscope (Leica Microsystems Ltd., Heerbrugg, Switzerland). Photographs of the host plant, damage and portable cases on the ground were taken using a Canon EOS 5D Mark IV camera plus a Canon micro 100 mm lens (Canon Inc., Tokyo, Japan). Photographs of genitalia and fine structures of larvae were taken with a Leica DM750 microscope (Leica Microsystems Ltd., Heerbrugg, Switzerland). All the photographs were typeset with Photoshop ® CS4 software (Adobe Systems, San Jose, CA, USA).
The genome of three larval specimens of the new species was extracted using Qiagen DNeasy Blood & Tissue Kit (Qiagen, Shildon, Germany), and the mitochondrial COI gene fragment was amplified using the primer pair HCO2198/LCO1490 [17]. The PCR system and the thermal cycling conditions followed deWaard et al. (2008) [18]. The PCR products obtained by amplification were sent to Qingke Biological Company, China, for sequencing. Thirty-one public barcode sequences, three of S. taigae and two of Adelidae as outgroups, were included in the analysis. All the sequences were deposited in a public dataset, DS-INCUR, in BOLD ( Table 1). The alignment of the new sequences and public ones and the genetic distance analysis were performed in MEGA X [19]. A maximum likelihood tree was created using MEGA X, and bootstrap values were calculated with 1000 replicates. Host plant family names were based on APG IV (2016) [20], and species names were based on The Plant List (2013) [21]. The morphological terminology of the adult moth was in accordance with Nielsen (1982) [10], and that of the immature stages followed Zimmerman (1978) [11,22].
Subclemensia taigae Kozlov, 1987 Diagnosis: Subclemensia is monotypic, so the species is diagnosed with species from related genera. Subclemensia taigae has white markings on its forewings, distinguishing it from all other species of the genus Paraclemensia. The male genitalia of this species are similar to those of P. oligospina Nielsen, 1982, a species known only by the male, but can be distinguished by the following features. In S. taigae, the posterior margin of the tegumen is nearly straight, the distal 2/3 of the phallus has a long ridge on the outer wall and a small triangular protrusion is present near the distal end, while in P. oligospina, the posterior margin of the tegumen has a markedly U-shaped indentation, and there is a long and curved spine protruding from the end of the phallus, without other protrusions (Nielsen 1982: Figures 22 and 24) [10]. The ovipositor of the female genitalia of S. taigae is most related to that of P. cyanea Nielsen, 1982, a species known only by the female (Nielsen 1982: Figure 37) [10], but can be distinguished from the latter by the following features. In S. taigae, the vestibulum has a pair of triangular sclerotized zones, and the ductus bursae and the corpus bursae are nearly equal in width, while in P. cyanea, the vestibulum is completely membranous, and the corpus bursae is significantly wider than the ductus bursae (Nielsen 1982: Figure 43) [10]. The forewing markings of S. taigae are similar to those of Incurvaria praelatella (Denis & Schiffermüller, 1775) [23] but can be distinguished by the following features. The new species has dark brown cilia around the apex of the forewings and is absent of a long spine process at the end of the phallus; in I. praelatella, the cilia at the apex of the forewings are white, and a long spine process is present at the end of the phallus (Kuprijanov 1994: Figures 4A and 8A) [24]. In addition, the forewing markings of S. taigae are similar to those of Alloclemensia unifasciata Nielsen, 1981 [15] and Phylloporia bistrigella (Haworth, 1828) [25], but there are significant differences in the male genitalia (Okamoto and Hirowatari 2004: Other materials. Eight larvae, mounted in slide, 2021.viii.14, ix.9, field no. LTT01116, LTT01117, LTT01119, other information same as adult. Adult (Figures 1 and 2). Wingspan 8-9 mm. Vertex orange yellow, frons yellowish white ( Figure 1e). Maxillary palpus 5 segments, pale yellow. Labial palpus 3 segments, half-length of maxillary palpus, pale yellow, tinged white distally. Proboscis reduced. Antennae 3/4 length of forewing, scape and pedicel yellowish white with golden luster, flagellum brown. Thorax and tegula brown with golden luster. Forewing blackish brown with golden luster at base, with bluish-purple metallic luster towards costa (Figure 1c,d); a transverse white fascia at basal 1/3, narrower at middle, tinged light yellow on costa and dorsum; two triangular white spots on costal 2/3 and on dorsum near tornus, tinged light yellow on wing margin, costal one slightly larger; a small white spot far before apex; cilia blackish brown, with golden luster. Hind wing gray, with bluish-purple metallic luster in disc and golden luster along margin; cilia gray, with golden luster, more prominent at base. Venation matches ground plan of the genus (Figure 2a). Leg black dorsally, white ventrally, hind tarsus white; without protibial epiphysis (Figure 2b). Abdomen dark brown dorsally, white ventrally.
Adult (Figures 1 and 2). Wingspan 8-9 mm. Vertex orange yellow, frons yellowish white (Figure 1e). Maxillary palpus 5 segments, pale yellow. Labial palpus 3 segments, half-length of maxillary palpus, pale yellow, tinged white distally. Proboscis reduced. Antennae 3/4 length of forewing, scape and pedicel yellowish white with golden luster, flagellum brown. Thorax and tegula brown with golden luster. Forewing blackish brown with golden luster at base, with bluish-purple metallic luster towards costa (Figure 1c,d); a transverse white fascia at basal 1/3, narrower at middle, tinged light yellow on costa and dorsum; two triangular white spots on costal 2/3 and on dorsum near tornus, tinged light yellow on wing margin, costal one slightly larger; a small white spot far before apex; cilia blackish brown, with golden luster. Hind wing gray, with bluish-purple metallic luster in disc and golden luster along margin; cilia gray, with golden luster, more prominent at base. Venation matches ground plan of the genus (Figure 2a). Leg black dorsally, white ventrally, hind tarsus white; without protibial epiphysis (Figure 2b). Abdomen dark brown dorsally, white ventrally.   Male genitalia (Figure 3). Tegumen trapezoidal to semicircular ( Figure 3a); a trapezoidal sclerotized zone on posterior margin of tegumen, with a long bristle laterally, a pair of small protrusions at middle each with a long bristle, a small sharp protrusion at middle ventrally (obvious on lateral view (Figure 3b,e)). Valva slightly rectangular, shorter than length of vinculum + saccus, widest nearly same as end of saccus; a small hump in middle and distal 1/3 of ventral margin, with 4 to 5 scale-like setae on each, an inconspicuous hump before distal end with some 10 scale-like setae around, narrower towards apex. Transtillae inverted U-shaped, in connection to base of valva by a hammer-like sclerotized band (Figure 3a). Vinculum + saccus nearly triangular (unrolling), anterior margin truncated. Phallus longer than valva, tubular, truncated at base, nearly equal width in middle, slightly wider distally; distal 2/3 with a long ridge on outer wall (Figure 3c), a small triangular protrusion at proximal end (Figure 3b), cornuti 7-8 sharp spines and numerous microspines towards base (Figure 3b). Juxta with a pair of sclerotized bands strongly narrowed and spiral on distal part, pointed distally; branches from middle of preceding bands joined to an arrow, with secondary branches from middle (Figure 3d).  (Figure 3). Tegumen trapezoidal to semicircular ( Figure 3a); a trapezoidal sclerotized zone on posterior margin of tegumen, with a long bristle laterally, a pair of small protrusions at middle each with a long bristle, a small sharp protrusion at middle ventrally (obvious on lateral view (Figure 3b,e)). Valva slightly rectangular, shorter than length of vinculum + saccus, widest nearly same as end of saccus; a small hump in middle and distal 1/3 of ventral margin, with 4 to 5 scale-like setae on each, an inconspicuous hump before distal end with some 10 scale-like setae around, narrower towards apex. Transtillae inverted U-shaped, in connection to base of valva by a hammer-like sclerotized band (Figure 3a). Vinculum + saccus nearly triangular (unrolling), anterior margin truncated. Phallus longer than valva, tubular, truncated at base, nearly equal width in middle, slightly wider distally; distal 2/3 with a long ridge on outer wall (Figure 3c), a small triangular protrusion at proximal end (Figure 3b), cornuti 7-8 sharp spines and numerous microspines towards base (Figure 3b). Juxta with a pair of sclerotized bands strongly narrowed and spiral on distal part, pointed distally; branches from middle of preceding bands joined to an arrow, with secondary branches from middle (Figure 3d).

First instar in case
Mature larva (Figure 6c-f). White with dark head and cervical shield. Body length 5.67-6.00 mm, width 0.87-1.17mm. Head capsule 0.60-0.65 mm in length, 0.75-0.80 mm in width. Dark spot from mesothorax to A8 along ventral meson line equal in size. Prolegs and crochets similar to early instars (Figure 6e,f).
Portable case (Figure 6g,h). Flat oval (Figure 6g). Single layer when first appears, double layers in mature stage. Inner layer smaller than outer, pale yellow, attached to outer layer by thin white mesh; larvae between inner layers. Outer layer composed of two pieces of leaf tissue in different sizes. Larvae feed in single-layered case in early stage, and some feces can be attached to outer surface of single-layered case. Outer layers can be formed by using leaf tissues in later instar; meanwhile, feces attached on outer surface of single-layered case can be covered by outer layer (Figure 6h).
Biology. Larvae live as leaf miners in the early stage (Figure 7a-c) and later construct a portable case from the wall of the mine and feed as leaf skeletonizers (Figure 7f). Early mines are yellowish-green spots (Figure 7a) and gradually become translucent over feeding (Figure 7b,c). Larvae can expel some fecal pellets out of the mine and attach them on the lower surface of the mine with silk threads (Figure 7c,d). The mines are expanded into irregular patches until forming single-layered cases forming oval holes on the leaves (Figure 7e). This species has one generation per year and overwinters as prepupae in a case. Emergence occurs in mid-May of the second year, and the earliest mine occurs in late May. The larvae grow very slowly during the leaf-mining stage. A significant increase in the size of the mine does not occur until mid-August. Not long after that, the larvae will make single-layered cases. In early September, the mature larvae make an outer case and fall onto the ground to find a place for overwintering (Figure 7g,h).

Discussion
Immature stages are poorly known in Incurvariidae, with half of the genera lacking any information on immature stages. Some immature morphological features are of taxonomic value [11]. Therefore, it is particularly useful to describe the immature stages of Incurvariidae for the identification of the immature stages. Some specific characters may also be useful for testing morphological synapomorphies and thus for stabilizing the generic nomenclature. The discovery of the immature stages of S. taigae enables us to compare the immature characters among species from closely related genera (Table 2).

A. unifasciata
Not available Outer layer composed of one piece of leaf tissue [8] Paired small sclerotization on lateral side of tergite of T2 [8] 1. Hind leg reaching posterior margin of A8; 2. A single row of spines on A3-8 [8].

P. acerifoliella
A broad corridor or a patch [12] Outer layer composed of one piece of leaf tissue (Pohl et al. 2015: Figure 13) [12], but can also be of two pieces Large sclerotization on T2 and T3 1. Hind leg beyond anterior margin of A8; 2. A single row of spines on A2-8 [11].
The cut-off of a leaf is one of the synapomorphies of Incurvariidae, but the initial mine by first-instar larvae differs among genera based on available data. The initial mine is a very short and irregular corridor in S. taigae and never forms a slender linear mine, which is quite different from the slender initial mine of A. mesospilella but somewhat similar to that of P. acerifoliella. The features on a leaf mine are of potential value for stabilizing the generic concept. A slender initial mine is extremely characteristic of A. mesospilella compared with other species of related genera, and it could be a generic character for Alloclemensia, but this cannot be confirmed further until at least the leaf mine of a second Alloclemensia species is known.
The sclerotized tergite of the prothorax is one of the larval synapomorphies of Incurvariidae; however, the degree of sclerotization on T2 and T3 varies among genera. In the type genus Incurvaria, the tergite of each thorax is usually sclerotized as well as the tergite of some anterior abdominal segments. Large areas of sclerotization are present on T2 and T3 in P. acerifoliella, but only a small sclerotized patch on each lateral side of T2 and T3 in A. mesospilella and A. unifasciata is found. No sclerotized zones are found on T2 and T3 in S. taigae. The status of the sclerotization on T2 and T3 seems discontinuous enough to separate Paraclemensia, Alloclemensia and Subclemensia. The absence of sclerotization on the tergite of T2 and T3 is probably a generic character for Subclemensia, while the small sclerotized patch on each lateral side of T2 and T3 is shared by species of Alloclemensia. However, more information on the immature stages of other species is necessary to test the hypothesis; then, the respective status of the sclerotization on T2 and T3 could be added to the generic concepts of relevant genera.
The portable cases of mature larvae are similar among genera, but the number of the leaf pieces comprising the outer layer varies between and within genera, and sometimes within an identical species, i.e., P. acerifoliella (Pohl et al., 2015) [12]. Therefore, it is still early to define a generic character using portable cases based on available data.
Pupae of Subclemensia, Alloclemensia and Paraclemensia share vestiges of mandibles, prominent maxillary and labial palpi, a single row of spines on A3-8 and two larger spines on A9, with the length of the antenna differing in males and females. It is difficult to trace a unique and reliable character for Subclemensia in pupae. Therefore, no reliable pupal character can be considered as a generic character according to current knowledge.