Three new species of Herpetogramma Lederer (Lepidoptera, Crambidae) from China

Abstract Five species of the genus Herpetogramma in China are studied with morphological and DNA barcode data. Herpetogrammabiconvexa Wan, Lu & Du, sp. nov., H.longispina Wan, Lu & Du, sp. nov., and H.brachyacantha Wan, Lu & Du, sp. nov. are described as new. Herpetogrammarudis (Warren) and H.magna (Butler) are newly diagnosed. Photographs of the habitus and genitalia of these five species are provided.


Introduction
The genus Herpetogramma was established by Lederer (1863) for the type species H. servalis Lederer, 1863. There are 103 species of Herpetogramma recorded in the Global Information System on Pyraloidea (Nuss et al. 2019). Twenty-two species were recorded in China before our study (Wang 1980;Wang and Speidel 2000;Du 2008;He 2014). This genus was well studied in North America (Solis 2010;Handfield and Handfield 2011;Scholtens and Solis 2015). In regions around China, 10 species were recorded in Korea (Bae et al. 2008;Kim et al. 2012;Roh et al. 2014;Park et al. 2016), 18 in Japan (Yamanaka 1960(Yamanaka , 1976Inoue 1982;Yamanaka 2003;Sasaki and Yamanaka 2013), two in Nepal and three in India (Yamanaka 1995;Mathew 2006). Most species found in these surrounding regions are distributed in China. The adults of most species are brown of various shades with distinct wing patterns, so that light brown, brown, and dark brown are used to describe three general major shades. The genitalia structures are very conservative, exhibiting only subtle differences among species (Shaffer and Munroe 1989;Du 2008Du , 2009Solis 2010Solis , 2011Handfield and Handfield 2011). Dissection is essential to identify species of this genus. In this paper, morphological and DNA barcode data were combined to identify three new species of Herpetogramma.

Taxon sampling
Specimens were collected by light trap and killed by ethylacetate or ammonium hydroxide. Genitalia preparation mainly followed the methods introduced by Li and Zheng (1996). Genitalia were examined and described before being mounted on microscope slides. The images of the adults were taken with a digital camera (Nikon P7700) and the images of the genitalia were prepared with a digital camera (Leica DFC 450) attached to a digital microscope (Leica M205 A).
Specimens examined, including types of new species, are deposited in the College of Plant Protection, Southwest University, Chongqing, China (SWUCPP) except for six specimens, including three paratypes, which are deposited in the Insect Collection of the College of Life Science, Nankai University, Tianjin, China (NKU). Among 80 sequences analyzed in the study, 12 sequences were downloaded from the BOLD database at http://v4.boldsystems.org/, two sequences were downloaded from GenBank and 66 newly obtained sequences were deposited in GenBank and can be accessed through the accession numbers listed in Table 1.

DNA extraction, PCR amplification, and sequencing
In total twelve species of Herpetogramma were included for PCR analysis and DNA sequencing (Table 1). Total DNA was extracted from legs of fresh or dry specimens using the TIANGEN DNA extraction kit following the manufacturer's instructions. The 658-base pair (bp) barcode region of COI was amplified with the LepF1/LepR1 primers (Hajibabaei et al. 2006). PCR products were sent to Sangon Biotechnology Co., Ltd. (Shanghai, China) for sequencing using the aforementioned primers.

Data analysis
All COI sequences were aligned by MUSCLE 3.8 and corrected by eye after being translated into amino acid sequences (Edgar 2004). Intraspecific and interspecific genetic divergence values were quantified based on the Kimura 2-parameter (K2P) distance model (Kimura 1980) and assessed by MEGA 6.0.6 (Tamura et al. 2013). Phylogenetic analysis was performed based on Maximum Likelihood (ML) with the GTR GAMMA model of nucleotide substitution, and with 1000 bootstrap replicates (Stamatakis et al. 2008). Syllepte invalidalis South, 1901 was chosen as the outgroup as it was never considered to be congeneric with Herpetogramma, but is part of the same subfamily (Spilomelinae).

DNA sequence analysis
A total of 77 COI sequences of Herpetogramma were analyzed. The dataset contained no obvious pseudogenes, supporting the assumption that the correct target gene sequence was amplified and sequenced. We observed 12 monophyletic clades for Herpetogramma in the resulting phylogenetic tree (Fig. 1). The pairwise genetic distances within and between these lineages are given in Table 2. Average intraspecific genetic distance ranged from 0.03 to  0.75% while average interspecific genetic distance ranged from 2.93 to 11.17%. The monophyla observed in the phylogenetic analysis were in full congruence with our morphological hypotheses for the investigated species, i.e., our morpho-species hypotheses are in accordance with the barcode clusters recovered (Fig. 1). Diagnosis. Frons rounded. Labial palpus obliquely upturned, porrect or upcurved; third segment short and blunt. Maxillary palpi filiform. Male antenna with ventral cilia. Forewing with length of cell ca. half of wing; R from cell at ca. four-fifths above; Rs 1 closely approximated to Rs 2 +Rs 3 ; Rs 4 curved toward Rs 2 +Rs 3 at base; M 2 , M 3 and CuA 1 from posterior angle of cell, nearly evenly spaced at base; CuA 2 from cell at fourfifths below. Hindwing with length of cell less than half of wing length; discocellulars arcuate curved; Rs anastomosed with Sc+R ca. basal one-fourth beyond cell; M 2 , M 3 and CuA 1 from posterior angle of cell, M 2 and M 3 approximated at base; CuA 2 from cell at two-thirds below (Fig. 2). Male genitalia: Uncus basiconic, narrow normally, with hairs distally; valva at base with small lamellate projection or clasper in some species. Female genitalia: Apophysis anterioris longer than apophysis posterioris; ductus bursae shorter than corpus bursae; corpus bursae with a sub-square signum.   (Warren, 1892), but can be distinguished from the latter by the dark brown wings tinged with pale yellow, forewing length 12.5-15.5 mm (wingspan 29.0-34.0 mm), finger-like projection on the posterior margin of the sacculus broad and with many tiny protrusions, and corpus bursae with central depression ca. half depth of diameter of the corpus burse. In H. rudis wings are light brown tinged with white, with a forewing length of 9.0-12.0 mm (a wingspan of 21.0-27.0 mm), the finger-like projection on the posterior margin of the sacculus is slender and without tiny protrusions, and the central depression of the corpus bursae is ca. 1/3 the depth of the diameter of the corpus burse.

Checklist of Herpetogramma species of China
Description. Adult (Figs 3, 4): Forewing length 12.5-15.5 mm (wingspan 29.0-34.0 mm). Frons rounded, yellowish brown. Vertex with erect brown scales, white close to eye. Antenna dark brown dorsally, yellowish brown ventrally; male antenna with ventral cilia ca. half as long as diameter of flagellomere. Labial palpus obliquely upturned, basal half white and distal half brown. Thorax and abdomen dark brown dorsally, silvery white ventrally. Legs yellowish white; fore tibia brown distally. Wings dark brown tinged with pale yellow, slightly darker in female. Forewing with orbicular spot and discoidal spot black, the latter reniform; antemedial line dark brown, slightly excurved, adjoined by a light-yellow line inside; postmedial line dark brown, from ca. 2/3 of costa, nearly straight to M 1 , excurved and serrated from M 1 to CuA 2 , then sharply incurved, and nearly vertical to inner margin below posterior angle of cell, adjoined by a light-yellow line outside. Hindwing with pattern of postmedial line similar to forewing; discoidal spot black. Cilia of wings brown, white along anal angle of hindwing. Abdomen long, second segment with two inconspicuous dark spots basally.  (Figs 13, 14). Uncus basiconic, distal half narrowed and bearing dorsal hairs, apex pointed and naked. Valva elongate lingulate, densely ciliated, costa slightly widened at middle, and with a small lamellate basal projection bearing hairs distally (Fig. 14A). Sacculus with a broad finger-like projection bearing many tiny protrusions at basal 2/3 of posterior margin, sparse long hairs on top of protrusions (Fig. 14B). Transtilla subtriangular, weakly sclerotized, meeting in middle. Juxta oval, a weakly sclerotized plate. Saccus developed, triangular, distinctly pointed distally. Phallus cylindrical, nearly same length as valva, without cornuti.
Etymology. The specific name, biconvexa, is derived from the Latin bi (meaning 'two' or 'double') and convexus (meaning 'arched outward'), in reference to the fingerlike projection bearing tiny protuberances on the posterior margin of the sacculus.

Remarks.
The identification of H. rudis was based on the description and photographs of external morphology and genitalia (Warren 1892;Inoue 1982;Bae et al. 2008;Sasaki and Yamanaka 2013). Although species of this genus have similar appearance and conservative genitalia, they can be differentiated according to their subtle and definite characteristics. In addition, it was found that the genitalia of H. biconvexa and H. rudis were different in size, but the ratios between structures were nearly the same, such as the length ratio between the uncus and the valva at ca. 1:4, ca. 4:3 between the uncus and the saccus, ca. 4:5 between the phallus and the transverse distance of the valva, and nearly 1:7 between the ductus bursae and the corpus bursae. We have not observed these characteristics in other similar species studied. Diagnosis. The species is similar to H. magna (Butler, 1879), but can be distinguished from the latter by its light brown wings, the broad uncus blunt at apex and elongate lingulate valva, corpus bursae sharply narrowed posteriorly, and the boundary indistinct between the ductus bursae and corpus bursae. In H. magna, wings are brown or dark brown; the narrowed uncus is pointed at apex and the valva is subfusiform, the corpus bursae is slightly narrowed posteriorly, and the boundary is distinct between the ductus bursae and corpus bursae.

Herpetogramma longispina
Description. Adult (Figs 7, 8). Forewing length 14.5-16.0 mm (wingspan 32.0-34.0 mm). Frons rounded, brown or light brown. Vertex with erect orange-yellow scales. Antenna light brown, male antenna with ventral cilia ca. half as long as diameter of flagellomere. Labial palpus obliquely upturned, basal 2/3 white and distal 1/3 light brown. Thorax and abdomen light brown dorsally, silvery white ventrally. Legs silvery white, fore tibia brown basally. Wings light brown tinged with faint yellow, lines and spots brown, distinct. Forewing with orbicular spot and reniform discoidal spot, faint yellow between orbicular spot and discoidal spot. Antemedial line excurved slightly, adjoined by a light-yellow wider line inside; postmedial line from ca. 2/3 of costa, straight to M 1 , excurved and pointedly serrated from M 1 to CuA 2 , then sharply incurved, and nearly vertical to inner margin below posterior angle of cell, adjoined by a wide and serrated light-yellow line outside. Hindwing with pattern of postmedial line similar to forewing, discoidal spot reniform. Cilia of wings brown, white along anal angle of hindwing.
Etymology. The specific name, longispina, is derived from the Latin longus (meaning 'long') and spina (meaning 'thorn'), in reference to a cluster of long spinose cornuti in male genitalia.
Distribution. China (Sichuan, Hubei).  (Butler). It can be distinguished from them by its wing pattern, which is not as distinct as those of the latter two species. In the male genitalia, its uncus is thinner than that of H. longispina and thicker than that of H. magna; its cornuti are the shortest, ca. 1/9 of the length of the phallus, while in the latter two species, they are ca. 1/4 of the phallus length. In the female genitalia, the corpus bursae are slightly narrowed posteriorly and the boundary is distinct between ductus bursae and corpus bursae in this species and H. magna, while the corpus bursae is sharply narrowed posteriorly and the boundary is indistinct between the ductus bursae and corpus bursae in H. longispina.

Herpetogramma brachyacantha
Description Adult (Figs 9, 10). Forewing length 13.5-15.5 mm (wingspan 31.0-35.0 mm). Frons rounded. Vertex with erect dark brown scales. Antenna brown, male antenna with ventral cilia ca. half as long as diameter of flagellomere. Labial palpus obliquely upturned, basal 2/3 white and distal 1/3 fuscous. Thorax and abdomen dark brown dorsally, silvery white ventrally. Legs silvery white, fore tibia brown distally. Wings brown tinged with faint yellow, lines and spots dark brown and slightly indistinct in female. Forewing with orbicular spot and reniform discoidal spot, faint yellow between orbicular spot and discoidal spot. Antemedial line indistinct, postmedial line from 2/3 of costa, straight to M 1 , excurved and pointedly serrated from M 1 to CuA 2 , then sharply incurved, and nearly vertical to inner margin below posterior angle of cell, adjoined by a serrated light-yellow line outside. Hindwing with pattern of postmedial line similar to forewing, discoidal spot reniform. Cilia of wings brown, white along anal angle of hindwing.
Etymology. The specific name, brachyacantha, is derived from the Greek words brachys (meaning 'short'), ake (meaning 'thorn') and anthos (meaning 'flower'), in reference to a cluster of short spinose cornuti in male genitalia.
Remarks. The identification of this species was based on the description and photographs of external morphology and genitalia (Butler 1879;Inoue 1982;Bae et al. 2008;Sasaki and Yamanaka 2013).

Discussion
Species of Herpetogramma are generally so similar in adult morphology and genitalia that a combined analysis of external morphology, genitalia structures and molecular data is strongly advised for their identification. The three new species described in this paper were discovered by integrating these three sources of data. Over the course of the last decade, an extensive number of specimens of this genus was collected by the members of our laboratory in most regions of China. Judging from preliminary research on these specimens, more new species and/or new records of this genus may be discovered in China in the future. We aim to collect more fresh specimens and obtain more molecular data of Herpetogramma species from China and intend to review the whole genus in the future.