Revision of Nagiella Munroe (Lepidoptera, Crambidae), with the description of a new species from China

Abstract The genus Nagiella was studied using morphological and DNA barcode data. Nagiella bispinasp. nov. is described as a new species, and N. hortulatoides Munroe is recorded in China for the first time. The diagnosis of this genus is revised, and the genitalia description of N. quadrimaculalis (Kollar and Redtenbacher) and N. inferior (Hampson) are given in English for the first time. Nosophora incomitata (Swinhoe) stat. rev. is removed from the synonym of N. quadrimaculalis. Photographs of the habitus and genitalia as well as COI DNA Barcode data of these four species are provided.

To date, four species of Nagiella have been identified worldwide, and they have been recorded in the Palaearctic and Oriental realms. These species are all distributed in China, with N. hortulatoides Munroe, 1976 being recorded in China for the first time in this study. Nagiella inferior and N. quadrimaculalis are widely distributed in the Palaearctic and Oriental realms (Wang 1980;Inoue 1982;Bae et al. 2008;Du 2009;Sasaki and Yamanaka 2013), with the latter species also recorded from Central Africa (Ghesquière 1942). In addition to China, N. hortulatoides is distributed in Myanmar. Nagiella occultalis Misbah & Yang in Ullah et al. 2017 is only distributed in China (Ullah et al. 2017). In this study, one new species, Nagiella bispina, is described based on morphological and DNA barcode data, and the diagnosis of this genus is revised.

Taxon sampling
The specimens were collected by light trap at night and killed by ethyl acetate or ammonium hydroxide. The specimens are deposited in the College of Plant Protection, Southwest University, Chongqing, China (SWUCPP) and the Institute of Zoology, Chinese Academy of Sciences, Beijing (IOZ). Information on the specimens from which the DNA Barcode region of the COI gene was sequenced is shown in Table 1. In total, 24 sequences were analysed in this study, with eight being from the BOLD database (Ratnasingham and Hebert 2007;http://v4.boldsystems.org/). The sequences obtained from our laboratory have been uploaded to BOLD.
Genitalia preparation mainly follows Li and Zheng (1996). Images of the adults were captured with a digital camera (Nikon P7700), and images of the genitalia were captured with a digital camera (Leica DFC 450) attached to a digital microscope (Leica M205 A).

DNA extraction, PCR amplification, and sequencing
In total, all five species of Nagiella were included for PCR analysis and DNA sequencing (Table 1). Total DNA from legs of fresh or dry specimens was extracted using the TIANGEN DNA Kit following the manufacturer's instructions, and the 658-base pair (bp) barcode region of COI was amplified using the LepF1/LepR1 primers (Hajiba-

Data analysis
All COI sequences were aligned by MEGA 7.0 (Kumar et al. 2016) and adjusted visually after being translated into amino acid sequences. Intraspecific and interspecific genetic divergence values were quantified based on the Kimura 2-parameter (K2P) distance model (Kimura 1980). 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). Patania balteata and P. chlorophanta were chosen as the outgroup species as they were members of the same tribe (Agroterini), but not congeneric with Nagiella.

DNA sequence analysis
Overall, 24 COI sequences, including six of the outgroup species, were analysed. The dataset contained no obvious pseudogenes, indicating the correct target gene sequence was amplified and sequenced.
Five monophyletic clades for Nagiella were observed in the resulting phylogenetic tree (Fig. 1). The pairwise genetic distances within and between these lineages are given in Table 2. The average intraspecific genetic distance ranged from 0.00 to 0.02%, while the average interspecific genetic distance ranged from 3.30 to 9.46%. The maximum intraspecific COI genetic distance was much less than the minimum interspecific distance. The monophyla observed in the phylogenetic analysis were in full congruence with our morphological hypotheses for the investigated species (Fig. 1).
Diagnosis. Frons rounded. Labial palpus broad, obliquely upturned and curved, compressed, third joint extremely minute, short and stout (Fig. 2). Male antenna with ventral cilia. Legs smooth. Fore wings near rectangular at the tips; length of cell ap- proximately half of wing; R from cell at approximately two-thirds; Rs 2 anastomosed with Rs 3 approximately three-fifths beyond cell; Rs 1 closely approximated to Rs 2 +Rs 3 ; Rs 4 curved towards Rs 2 +Rs 3 at base; discocellulars arcuately incurved; M 2 , M 3 and CuA 1 from posterior angle of the cell uniformly at the base; CuA 2 from three-fourths below the cell. Hindwing with length of cell one-third of wing; Sc+R anastomosed with Rs approximately one-fourth beyond the cell; M 2 , M 3 and CuA 1 separately from posterior angle of the cell; CuA 2 from two-thirds below the cell; discocellulars incurved ( Fig. 3).
Male genitalia: Uncus short and wide; gnathos present in most species; valva lingulate, posterior margin with long setae cluster in most species; clasper near base, developed and pointed to sacculus; phallus cylindrical, cornuti absent in most species. Female genitalia: Apophyses anteriores longer than apophyses posteriores, rhomboidally expanded near base; ductus seminalis from the ductus bursae; corpus bursae oval, with signum. Remarks. According to Munroe (1976) and Ullah et al. (2017), Nagiella can be differentiated from its similar genera by its short and wide uncus, developed gnathos, broader valva with stout setae subapically, large oblique clasper and absence of cornuti, as well as by the type of wing maculation. In N. bispina sp. nov., however, the gnathos is absent, the valva costa has no stout setae, and cornuti are present. Other morphological and DNA barcode data of this new species indicate it as a member of the genus. Therefore, the diagnosis of Nagiella was revised based on previous studies and our research, including supplementation of the wing venation.
Key to species of Nagiella based on morphology and genitalia  . 4): Frons, palpi, basal antenna, most of vertex black. Thorax orange with blackish-fuscous spot. Wings white, light orange at base, maculation grey, with terminal line white, discontinuous. Forewing with orbicular spot and discoidal spot round, a large elongate elliptical spot from base to orbicular spot below cell; grey terminal area broad, with inside concave between M 1 and CuA 2 . Hindwing with discoidal spot round; grey terminal area broad, with inside slightly concave between M 2 and CuA 2 . Abdomen with first and second segment orange with three black spots, the rest grey. Male genitalia (Fig. 9): Uncus trapezoidal. Gnathos slender, finger-like. Valve elongate lingulate, posterior margin with clusters of long setae near middle and terminal, clasper thickly finger-like. Female genitalia: Corpus bursae with a round signum (Munroe 1976).
Female genitalia (Fig. 13). Apophyses anteriores ca. twice the length of apophyses posteriores. Ductus bursae ca. twice the length of corpus bursae; ductus seminalis from the middle of ductus bursae. Corpus bursae oval, with a very small leaflike signum.
Female genitalia (Fig. 14). Apophyses anteriores ca. twice the length of apophyses posteriores. Ductus bursae ca. twice the length of corpus bursae, distinctly narrowed near the base; ductus seminalis from approximately one third of the ductus bursae. Corpus bursae oval, with a small round signum.
Remarks. In addition to Rhus chinensis Mill, another host, Metaplexis japonica Makino (Apocynaceae), was recorded by Fan and Piao (2013) in the same article according to Yoshiyasu (1991). However, we found M. japonica was recorded by Yoshiyasu (1991) as the host of Glyphodes quadrimaculalis (Bremer and Grey 1853) but not of N. quadrimaculalis (Kollar and Redtenbacher). Rhus chinensis Mill is the only host of N. quadrimaculalis (Kollar and Redtenbacher) known so far. Swinhoe (1894) stated that Nagia incomitata was between Nagia quadrimaculalis and N. flavispila, but quite different to either. But N. incomitata was regarded as a synonym of N. quadrimaculalis because they were similar in habitus (Bae et al. 2008). We investigated the original description and type specimen of N. incomitata Swinhoe, 1894, and compared them with the description and photographs of N. quadrimaculalis (Kollar and Redtenbacher 1844;Du 2009;Sasaki and Yamanaka 2013). The third segment of labial palpus of the former is slender and pointed distally (Fig. 7A), the forewing has no small white spot between the orbicular spot and discoidal spot, and the large white spot beyond the cell is down to the CuA 2 (Fig. 7); while the third segment of labial palpus of the latter is stubby and blunt distally (Fig. 2), the forewing has a small white spot between the orbicular spot and discoidal spot, and the large white spot beyond the cell is down to the CuA 1 (Fig. 6). Therefore, N. incomitata is not a synonym of N. quadrimaculalis. Nagia incomitata was transferred to Chalcidoptera Butler, 1887 by Swinhoe (1901) after stating previously that it did not belong into Nagia (Swinhoe 1900). Hampson (1896), on the other hand, considered it a synonym of Nosophora chironalis (Walker, 1859), which he later revised (Hampson 1903) by reinstating it as Nosophora incomitata, with the junior synonym Nosophora triguttalis Warren, 1896. In the same publication on page 216, Hampson (1903) synonymised the males of N. incomitata with Sylepta [sic] quadrimaculalis. For the time being (i.e., until the type material has been investigated), we conclude as Hampson (1903), Klima (1939b), and Mandal and Bhattacharya (1980), who considered incomitata a species of Nosophora.
Distribution. China (Shaanxi, Hubei) (Ullah et al. 2017). Diagnosis. This species is very similar to N. quadrimaculalis externally, but can be distinguished from the latter by its rather short and wide uncus with distal margin round, gnathos absent, clasper thick thorn-like, phallus with a hook-shaped cornutus; ductus bursae ca. the same length as corpus bursae with two thorn-like signa (Fig. 15A). In N. quadrimaculalis, the uncus is trapezoidal, gnathos is slender and finger-like, clasper is thickly finger-like, and phallus exhibits no cornuti; ductus bursae is ca. twice the length of corpus bursae and corpus bursae has a small round signum (Fig. 14).
Description. Adult (Fig. 8). Body brown tinged with copper-colour. Forewing length 11.5-13.5 mm (wingspan 26.0-30.0 mm). Frons, vertex, antenna and maxillary palpus brown. Male antenna with ventral cilia ca. half as long as the diameter of flagellomere. Labial palpus with first and second segments white ventrally, the rest brown. Thorax and abdomen brown dorsally, off-white ventrally. Legs off-white, fore tibia brown distally. Wings brown. Forewing with antemedial line excurved, unclear; orbicular spot and discoidal spot dark brown, the latter squarish; a small white spot between the orbicular spot and discoidal spot; a large white sub-reniform spot between the discoidal spot and postmedial line, up to Rs 2 +Rs 3 and down to CuA 1 ; postmedial line unclear, from ca. 2/3 of the costa, along outer edge of the large white spot, excurved from M 2 to CuA 2 , then incurved and nearly vertical to the inner margin below the posterior angle of cell; cilia lightly brown with white basal line. Hindwing with discoidal spot dark brown, short band; a large white irregular quadrilateral spot between the discoidal spot and postmedial line, dentated between M 2 and M 3 ; postmedial line unclear, along outer edge of the large white spot, lightly excurved from M 2 to CuA 2 , then incurved and nearly vertical to the inner margin below the posterior angle of cell; cilia lightly brown with white basal line. Abdomen with each segment white distally.
Etymology. The specific name, bispina, is derived from the Latin bi (meaning two or double) and spina (meaning spine or thorn) in reference to the two thorn-like signa.