The leaf-mining genus Antispila Hübner, 1825 feeding on Vitaceae in Shandong Peninsula, China with one new species (Lepidoptera, Heliozelidae)

Abstract The Antispila species feeding on Vitaceae from Shandong Peninsula, China are treated. Antispila kunyuensis Liu, sp. n., feeding on Ampelopsis humulifolia, is described as new to science, and A. uenoi Kuroko, 1987, feeding on Vitis amurensis, is recorded as new for China. Vitis amurensis is documented as a new host plant for A. uenoi. The adult male and female, host plant and typical patterns of leaf-mines of both species are illustrated, as are male and female genitalia and venation. The venation and the paired tufts of scales on the 7th abdominal segment in male are illustrated for A. uenoi for the first time. DNA barcodes of both species are also provided, together with a neighbor-joining tree for facilitating species delimitation.


Introduction
The family Heliozelidae comprises 126 described species in 12 genera (van Nieukerken et al. 2011, 2012, van Nieukerken and Geertsema 2015, with the largest diversity in North America and Australia. The Heliozelidae were only recently recorded as new for China, with one formally published species , but also several database records in BOLD (http://www.boldsystems.org). However, the knowledge of Chinese Heliozelidae is slowly increasing, and two more species feeding on Vitaceae are described in the present paper. Vitaceae comprises an important group of hosts for Heliozelidae, especially for the genus Antispila Hübner, 1825 (van Nieukerken et al. 2012). Eleven out of the 20 Palearctic and Oriental species of Antispila feed on Vitaceae (Meyrick 1926, Kuroko 1961, 1987, van Nieukerken et al. 2012, van Nieukerken and Geertsema 2015.
Shandong Peninsula is located in the east of China, facing the Korean Peninsula across the Yellow Sea. Although located in a relatively developed region, the arthropod diversity is still rather poorly known and lacks systematic work, so new species, especially small-sized ones, can still be discovered. Here, we discovered two species of the leaf-mining genus Antispila during an ongoing biodiversity exploration in Shandong Peninsula. One of these is a new species, the other one, A. uenoi Kuroko, 1987 is new for China. Both are described here in detail, increasing the number of known species of the genus Antispila in China to three.

Material and methods
Leaves with active mines were placed in small plastic bags for rearing. After the shields had been exscinded and the larvae had left the mines, leaves with vacant leaf-mines were dried in a plant press. The larval shields, the corresponding adults, and the vacant leaf-mines were identically coded.
Genitalia and wings were dissected and mounted according to the methods introduced by Li (2002), but stained with Eosin Y and/or Chlorazol Black. Illustrations were prepared by using a Leica DM1000 microscope. Adult photographs were taken with a Leica S6D stereo microscope. Photographs of the host plants and leaf-mines were taken in the field using a Canon EOS 650D camera plus a Macro Lens, and enlarged photographs of leaf-mines were taken with the Leica S6D stereo microscope.
DNA was extracted from adult specimens preserved in 95% ethanol in Shandong Normal University, Jinan, China, with the whole body skeleton including genitalia and wings preserved as vouchers (Knölke et al. 2005). Protocols for total DNA extraction and mitochondrial COI gene amplification followed that described in our previous study . The sequence data used in this study have been deposited in GenBank and in the BOLD database, a public dataset "DS-ANTIVIT" (https://doi.org/10.5883/ DS-ANTIVIT). Sequences were aligned using the MUSCLE model and genetic distance estimation was analyzed using the Kimura 2-Parameter model in BOLD.
Terminology for adults follows van Nieukerken et al. (2012); the term canalis spiralis of female genitalia follows Kuroko (1987). The classification of the host plants is based on APG (2016), and plant scientific names follow The Plant List (2013).
All the specimens examined, including the holotype of the new species, are deposited in the Zoological Collection of Shandong Normal University (SDNU). The type depository of A. uenoi Kuroko, 1987, collection of the Entomological Laboratory, University of Osaka Prefecture, is abbreviated as UOP. Diagnosis. Two Antispila species, A. ampelopsia Kuroko, 1961 andA. orbiculella Kuroko, 1961, are known to feed on Ampelopsis, and both associate with the same species, A. brevipedunculata. Antispila kunyuensis can easily be distinguished from A. ampelopsia by the fine features of the phallus and the ovipositor, and from A. orbiculella by the two separate basal spots that are joined forming a transverse fascia in the forewing.
Venation (Fig. 9). Forewing with Sc reaching before middle on costa; R 1 from 2/5 on upper margin of cell to costal 3/5, Rs 1 from distal 1/7 on upper margin of cell to costal 3/4, Rs 2 from beyond distal end of cell, Rs 3+4 reaching costa before apex; cell tri- and M 3 to dorsum; Cu to middle of dorsum; A 1+2 weak. Male with one long frenulum, female bearing two shorter frenular bristles.
Male genitalia (Figs 11-15). Tuba analis developed (Fig. 12). Uncus bar-shaped, protruded towards posteriorly at middle. Vinculum shorter than phallus, slightly rectangular on anterior margin. Valva more or less triangular, digital process long and narrow, almost same length as valva, pecten on pedicel, with nine comb teeth (Fig. 13). Juxta half as long as phallus, densely covered with small teeth on basal 2/5, anterior arrow pointed on basal corners. Phallus nearly as long as length of vinculum + tegumen, narrowed anteriorly (Fig. 11); phallotheca with five to six strong teeth and a group of smaller sharp-pointed teeth, with a cluster of smaller spines at base of juxta; distal part with a mushroom-shaped process and a straight process ventrally (Figs 14, 15).
DNA barcode. One DNA barcode from a paratype was obtained. A neighborjoining tree, covering most Asian Antispila species and other Vitaceae-feeding species, was generated for facilitating species delimitation (Fig. 33).   (Figs 34, 35). The placement of the leaf-mines is variable (Figs 36-38), from the base to the apex of a leaf, from absolutely along veins to between but not touching veins. This results in variably-shaped blotch mines, but the majority of blotches are more or less round (Figs 38,(42)(43)(44)(45). Frass primarily occupies the opposite side of the cut-out in round blotch mines, or occasionally disperses throughout the mine (Fig. 37), but always in a broad medial band in wide gallery mines (Fig. 38). This species overwinters as a prepupa in the shield. A single generation per year was observed at the type locality.
Female with forewing patterns more distinct (Figs 4, 5). Venation (Fig. 10). Forewing with Sc reaching before middle on costa; R 1 from 2/5 on upper margin of cell to costal 3/5, Rs 1 from distal 1/7 on upper margin of cell to costal 3/4, Rs 2 from well beyond distal end of cell, Rs 3+4 reaching costa before apex; cell  truncated distally; M 1 stalked with Rs 3+4 , to termen near apex, M 2+3 from lower corner of distal end of cell; CuA from distal 1/7 of lower margin of cell; A 1+2 to beyond middle of dorsum. Hindwing with Sc to beyond middle of costa, R+M ending in 4 branches: Rs to costa, M 1 to dorsum near apex, M 2 and M 3 to dorsum; Cu to middle of dorsum; A 1+2 weak. Male with one long frenulum, female bearing two shorter frenular bristles.
Male genitalia (Figs 16-25). Tuba analis developed. Uncus bar-shaped, with two papillae bearing two short setae each at middle, bearing one long and a few shorter setae laterally (Fig. 22). Vinculum shorter than phallus, rounded on anterior margin. Valva semicircular on ventral margin, digital process about half the width of valva, pecten with 12 comb teeth (Figs 20,21). Juxta longer than half length of phallus, anterior arrow broad and almost semicircular. Phallus as long as length of vinculum + tegumen, narrowed anteriorly; phallotheca with groups of spines, more concentrated and larger ventrally (Figs 24,25); distal part with two processes ventrally, one large and curved, the other Vshaped with one branch larger than the other, two smaller similar processes dorso-apically, one less sclerotized and curved process at apex with several membranous teeth ventrally (Fig. 23). Paired tufts of slender scales on the 7 th abdominal segment (Figs 8,17,18).
DNA barcode. Two DNA barcodes were obtained (Fig. 33). A partial DNA barcode of 268 bp generated from a paratype of A. uenoi (RMNH.INS.24531) was used for identification of the Chinese specimens. The genetic distance between the Chinese specimens and the paratype is 1.53%.  Host plants. Vitis amurensis Rupr. (Fig. 39), V. coignetiae Pulliat ex Planch. and V. labruscana L.H. Bailey (Kuroko 1987). Vitis amurensis is recorded here as a new host.
Biology. Leaf-mines on Vitis amurensis can occupy serrations along the leaf margin (Figs 40,41,(46)(47)(48) or the leaf basal area (Fig. 49) in an almost equal proportion, calculated from our rearing data (5 : 4); no other placements (e. g. leaf central area) of mines were observed, although in Japan the majority of mines occupy the apical or marginal area of the leaves on other hosts (Kuroko 1987). Frass often dispersed along mines (Figs 47-49). This species overwinters as a prepupa in the shield. Two generations probably occur in Shandong Peninsula.
Distribution. China (Shandong), Japan: Honshu. The host plant Vitis amurensis is widespread in the northeast and eastern parts of China (Chen et al. 2007), Eastern Russia (Afonin et al. 2008), Japan: Honshu and Korea (Ohwi 1965). A much wider distribution of the moth is expected, where its host plants occur.
Remarks. This species is newly recorded in China. The venation and the paired tufts of scales on the 7 th abdominal segment in male are illustrated for A. uenoi for the first time.

Discussion
In the Miocene, the arthropod diversity of Shandong Peninsula was quite rich, which is well documented by numerous fossil records from Shanwang, Shandong province (Zhang 1989(Zhang , 1994. During the last centuries, the Yellow River shifted its mouth a number of times and finally diverted to the Bohai Sea in 1855 (Cheng andXue 1997, Wang et al. 2010). Both long-term historical and recent regional processes may have significantly influenced the species richness of Shandong Peninsula, which makes this peninsula an interesting region for biogeographical and biodiversity studies (e.g., Zheng et al. 2009). Heliozelidae, together with other small-sized Lepidoptera species, are expected to experience an increase in species richness upon a deeper exploration of this region, as will be the case for all of China. For instance, in Argyresthiidae, the number of species increased from 14 to 64 after the study by . This would be true when considering the host preference of the heliozelids and the rich diversity of Vitaceae and many other host families in China. Take Vitaceae for example. A majority of Antispila species show a specific host preferences for Vitaceae and Cornaceae (Milla et al. 2017, van Nieukerken et al. 2018, and there are 146 species of Vitaceae distributed in eight genera in China, with 87 endemic species, which are mostly native to central, south, and southwest China (Chen et al. 2007).