Research Article
Research Article
A new cryptic species of Nagiella Munroe from China revealed by DNA barcodes and morphological evidence (Lepidoptera, Crambidae, Spilomelinae)
expand article infoMisbah Ullah§, Zhaofu Yang§, Pingping Qiao§, Yalin Zhang§
‡ Ministry of Education, Yangling, China
§ Northwest A&F University, Yangling, China
Open Access


Nagiella occultalis Misbah & Yang, sp. n. from China is described and illustrated. This new species is very similar to N. quadrimaculalis (Kollar, 1844) in general morphological characters of forewing and male genitalia. Molecular evidence shows that these two species diverge in COI barcode region by more than 3.2%. Sequence divergence among the two species is congruent with subtle morphological differences. Wing venation and male genitalia of the two species are compared and illustrated.


COI gene, genitalia, Scopula quadrimaculalis, taxonomy


The subfamily Spilomelinae (Crambidae) is the largest subfamily of pyraloid moths including about 3300 species in more than 300 genera having worldwide distribution (Munroe and Solis 1999). The genus Nagiella Munroe, 1976 is one of the less speciose genera of Spilomelinae (Munroe 1976). Compared to other genera of this subfamily and despite its small size, Nagiella has been little studied and no comprehensive studies have been made on the taxonomy of its constituent species. The only taxonomic efforts were made by Munroe in 1976. This genus was originally described as Nagia by Walker in 1866 based on the type species Nagia desmialis Walker, 1866. Munroe (1976) recognized that Nagia Walker, 1866 is a junior homonym of Nagia Walker, 1858 (Lepidoptera: Noctuidae) and replaced it with the new name Nagiella Munroe, 1976. This genus is widely distributed in Malaysia (Borneo and Sarawak), Burma, China, and Japan (Munroe 1976; Inoue 1982; Wang 1980). The genus comprises three described species: Nagiella inferior (Hampson, 1898), Nagiella quadrimacualis (Kollar, 1844) with two junior subjective synonyms, desmialis Walker, 1866 and incomitata Swinhoe, 1894, and Nagiella hortulatoides Munroe, 1976 distributed in northeastern Burma. The generic characters as defined by Munroe (1976) are: uncus truncate, short and wide; gnathos ribbon-like; subscaphium elongate; valva broader with stout setae subapically, sella digitiform, elongate and sharp; cornutus absent. This provides the baseline description of the genus on which the present study is based.

Recently the integration of DNA barcoding and morphological approaches opened the field for researchers in accelerating species identification and assisted in detecting previously undetected cryptic species (Sutrisno 2005; Mutanen et al. 2012; Haines & Rubinoff 2012; Yang et al. 2012; Rajaeish et al. 2013; Yang et al. 2016; Mally et al. 2016). The taxonomic placement of N. occultalis sp. n. has been unclear; therefore, an integrative approach was designed to study the generic differences (Munroe 1976). In the present integrative taxonomic study, N. occultalis sp. n. collected from Shaanxi and Hubei Province, China, is described.

Materials and methods

Taxon sampling

Three specimens of N. occultalis sp. n. were collected from Taibai Mountain, Shaanxi and Wufeng, Hubei in China and 15 specimens of N. quadrimaculalis were collected from various localities (Table 1). Genitalia preparation mainly follows Landry (2007) and Yang et al. (2012) and terminology follows Kristensen (2003). The images of adults and genitalia were captured with a Canon Power Shot SX60 digital camera and (ZEISS Discovery V20) stereomicroscope equipped with an AxioCam ICc5 camera, respectively and measurement was taken in mm by scale bar equipped in stereomicroscope. Type material of the new species is deposited in the Entomological Museum, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China (NWAFU).

Specimens of two Nagiella species from China examined in this study.

Identification BIN Process ID Sample ID Length of sequence (bp) GenBank Accession Province Genitalia slide number
N. occultalis sp. n. BOLD:AAD8179 CNPYB439-16 NAFU Pyr002290 658 KY080696 Shaanxi
N. occultalis sp. n. BOLD:AAD8179 CNPYB407-16 NAFU Pyr002397 658 KY080703 Shaanxi NAFU Pyr002065
N. occultalis sp. n. BOLD:AAD8179 CNPYD499-10 Pyr000499 658 HM908668 Hubei
N. quadrimaculalis CNPYA401-10 NAFU Pyr000401 0 Yunnan
N. quadrimaculalis CNPYA402-10 NAFU Pyr000402 0 Sichuan
N. quadrimaculalis CNPYA403-10 NAFU Pyr000403 0 Yunnan
N. quadrimaculalis CNPYA404-10 NAFU Pyr000404 0 Yunnan
N. quadrimaculalis CNPYB409-16 NAFU Pyr002070 0 Shaanxi NAFU Pyr002070
N. quadrimaculalis CNPYB410-16 NAFU Pyr002261 0 Shaanxi NAFU Pyr002261
N. quadrimaculalis CNPYB411-16 NAFU Pyr002262 0 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB412-16 NAFU Pyr002263 658 KY080700 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB413-16 NAFU Pyr002264 658 KY080702 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB414-16 NAFU Pyr002265 658 KY080704 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB415-16 NAFU Pyr002266 658 KY080698 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB416-16 NAFU Pyr002267 658 KY080694 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB417-16 NAFU Pyr002268 658 KY080705 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB418-16 NAFU Pyr002269 658 KY080697 Shaanxi
N. quadrimaculalis CNPYB419-16 NAFU Pyr002270 0 Shaanxi
N. quadrimaculalis CNPYB420-16 NAFU Pyr002271 0 Shaanxi
N. quadrimaculalis CNPYB421-16 NAFU Pyr002272 0 Henan NAFU Pyr002272
N. quadrimaculalis CNPYB422-16 NAFU Pyr002273 0 Henan NAFU Pyr002273
N. quadrimaculalis CNPYB423-16 NAFU Pyr002274 0
N. quadrimaculalis CNPYB424-16 NAFU Pyr002275 0 Hunan
N. quadrimaculalis CNPYB425-16 NAFU Pyr002276 0 Hunan
N. quadrimaculalis CNPYB426-16 NAFU Pyr002277 0
N. quadrimaculalis CNPYB427-16 NAFU Pyr002278 0 Fujian
N. quadrimaculalis CNPYB428-16 NAFU Pyr002279 0 Hainan
N. quadrimaculalis CNPYB429-16 NAFU Pyr002280 0 Hainan
N. quadrimaculalis CNPYB430-16 NAFU Pyr002281 0
N. quadrimaculalis CNPYB431-16 NAFU Pyr002282 0 Zhejiang
N. quadrimaculalis CNPYB432-16 NAFU Pyr002283 0 Yunnan
N. quadrimaculalis CNPYB433-16 NAFU Pyr002284 0 NAFU Pyr002284
N. quadrimaculalis CNPYB434-16 NAFU Pyr002285 0
N. quadrimaculalis CNPYB435-16 NAFU Pyr002286 0
N. quadrimaculalis CNPYB436-16 NAFU Pyr002287 0 NAFU Pyr002287
N. quadrimaculalis CNPYB437-16 NAFU Pyr002288 0
N. quadrimaculalis BOLD:AAD8178 CNPYB438-16 NAFU Pyr002289 658 KY080695 Shaanxi
N. quadrimaculalis BOLD:AAD8178 CNPYB440-16 NAFU Pyr002291 658 KY080701 Shaanxi NAFU Pyr002291
N. quadrimaculalis BOLD:AAD8178 CNPYB441-16 NAFU Pyr002292 658 KY080699 Shaanxi
N. quadrimaculalis CNPYB408-16 NAFU Pyr002398 0 Shaanxi NAFU Pyr002067
N. quadrimaculalis BOLD:AAD8178 CNPYD497-10 Pyr000497 622 HM908666 Hubei
N. quadrimaculalis BOLD:AAD8178 CNPYD498-10 Pyr000498 658 HM908667 Hubei
N. quadrimaculalis CNPYD500-10 Pyr000500 0 Hubei
N. quadrimaculalis CNPYD501-10 Pyr000501 0 Hubei
N. quadrimaculalis CNPYD502-10 Pyr000502 0 Hubei
N. quadrimaculalis BOLD:AAD8178 CNPYD503-10 Pyr000503 658 HM908669 Hubei
N. quadrimaculalis BOLD:AAD8178 CNPYD504-10 Pyr000504 658 HM908670 Sichuan
N. quadrimaculalis BOLD:AAD8178 CNPYD505-10 Pyr000505 658 HM908671 Sichuan

Kimura 2-parameter genetic distances calculated within (in italic) and between three species of Nagiella.

Nagiella occultalis sp. n. Nagiella quadrimaculalis Nagiella inferior Patania ruralis (outgroup)
Nagiella occultalis sp. n. 0.0000000 0.0072358 0.0086344
Nagiella quadrimaculalis 0.0320975 0.000787822 0.0101216
Nagiella inferior 0.0475427 0.0598071 0.000761036
Patania ruralis (Outgroup) 0.1156349 0.1165689 0.1134248 0.009202714

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from insect legs by following the method of Ivanova et al. (2006). PCR amplifications were conducted to amplify a full-length (658 bp) barcode region of the mitochondrial COI gene by the primers pairs, LepF1 and LepR1 (Hajibabaei et al. (2006). After the PCR products were checked with 1% agarose gel, sequencing was performed at Sangon Biotechnology Co., Ltd. (Shanghai, China) using the same primers as in PCR.

Data analysis

Sequence alignment was carried out by using MUSCLE algorithm implemented in MEGA 6.0 (Tamura et al. 2013). MEGA 6.0 was also used to perform genetic distances under the Kimura 2-parameter model of base substitution, to produce the Neighbor-Joining (NJ) tree, and to perform bootstrap analysis (1000 replicates) (Kimura 1980). In the present study, we included four sequences of Nagiella inferior and selected Patania ruralis (Scopoli, 1763) as the primary out-group to build the tree which is most closely related genus. Sequences obtained from the current study were deposited in GenBank, in addition to being available in the BOLD dataset DS-PLEQUA.


DNA sequence analysis

A total of 18 COI gene sequences of N. occultalis sp. n. and N. quadrimaculalis were obtained. The lengths were from 622–658 bp (mean 656 bp). The genetic distances within and between these two species of Nagiella are given in Table 2. Intraspecific genetic divergences ranged from 0.00–0.16 % (mean 0.078 %), whereas interspecific genetic divergence ranged from 3.12–3.28 % (mean 3.21 %). The neighbor-joining (NJ) tree (Fig. 1) showed two distinct barcode clusters that correspond to morphological differences between these two species.


Nagiella occultalis Misbah & Yang, sp. n.


The specific epithet refers to “cryptic”, as this previously undetected species stood within the N. quadrimaculalis complex.


This species can be distinguished from N. quadrimaculalis by the width and length of the uncus, the proportions of the valva and transtilla, and size of the forewing, as described in Table 3.


(Figs 2A, 3). Body yellowish brown to black with white patches on wings. Length of forewing 15–16 mm. Head with frons shiny white, labial palpus bent over top of head. Patagium shiny black. Forewing dark brown, with small bean-shaped white spot of varying size near middle of reniform stigma in the base of discal cell; rectangular subdiscal white spot proportionally narrower or elongate. R1 arising from cell at about apical third and almost parallel to Sc, R2 parallel to R1 but close to R3+4. R3 and R4 long stalked and reached apical margin. M2 and M3 closer to each other at base than M1 (almost of the same length) but all median veins on equal distance on outer margin. Vein Cu2 originating from 2/3 of the cell. Anal vein A1+2 prominent and complete while A3 diminished before mid-length of wing. Hind wing with bean-shaped white spots near outer margin of medial line at terminal part of discal cell; Sc, radial and M1 on same stalk, anal vein A3 incomplete.

Male genitalia (Fig. 4A, B). Uncus subtrapezoid in outline, posterolateral angles rounded, distal margin slightly notched medially. Gnathos with proximal arms extended transversely from teguminal margin and joined mesially into subclavate distal projection extended almost to level of apex of uncus. Subscaphium very elongate, apex extended beyond apex of valvae. Transtilla triangular, broad basally and apically narrower. Valva relatively short and broad with several thickened setae on posterior margin. Sella elongate, digitiform, straight laterally, apex rounded. Saccus roundly conical. Phallus cylindrical, terminal end somewhat tapered, cornutus absent.

Female. Unknown


China (Taibai Mountain, Shaanxi; Wufeng, Hubei).

Type material

Holotype. ♂: China: Shaanxi, Taibai Mountain, 1051 m, 25 July 2014, Zhou Lin (NWAFU), Specimen ID: NAFU PYR002397. Genitalia slide number: NAFU PYR002397. Paratypes. 1 ♂, same data as the holotype except 24 July 2014; 1 ♂, China, Hubei, Wufeng, Changleping town, 14 July 2008, Zhao Lu.


The genus Nagiella, formerly comprised of three recognized species widespread in Burma, China, Japan and Malaysia (Borneo and Sarawak), is now increased to four with N. occultalis sp. n.

Figure 1. 

Neighbor-joining tree (K2P) based on the 22 COI sequences of the three Nagiella species from China, rooted with Patania ruralis as outgroup. Bootstrap values <75 are not shown.

Figure 2. 

Adults, dorsal aspect A N. occultalis sp. n. B N. quadrimaculalis.

Figure 3. 

Wing venation of N. occultalis sp. n.

Morphological differences between Nagiella occultalis sp. n. and N. qudrimaculalis.

Characteristics N. occultalis sp. n. N. quadrimaculalis
Forewing length 15–16 mm (Fig. 2A) 18–20 mm (Fig. 2B)
Small subdiscal spot on forewing Proportionally narrower or elongate Sub-quadrate
Uncus width and length 0.4 × 0.6 mm (Fig. 4A) 0.3 × 0.68 mm (Fig. 4C)
Posterior margin of uncus Slightly notched medially Evenly rounded
Valva Broader, W/L 0.91 × 3.09 mm Slender, W/L 0.7 mm × 2.08 mm
Sella with ventral edge Straight Slightly incurved
Subscaphium Elongate, conical sclerotized Unsclerotized
Size of transtilla Narrower, 0.28 × 0.8 mm Broadly triangular 0.3 × 0.9 mm
Phallus Phallus L/valva L ratio 1.19 (Fig. 4B) Phallus L/valva L ratio 1.7 (Fig. 4D)
Figure 4. 

Male genitalia A, B N. occultalis sp. n., genitalia slide NAFU PYR 002397 C, D N. quadrimaculalis, genitalia slide NAFU PYR 002069.


Munroe (1976) indicated that Nagiella differs from Pleuroptya Meyrick, 1890 in several genital characters, i.e. short, wide uncus, gnathos developed, cornutus absent, valva broader with stout setae subapically, as well as in type of wing maculation. This taxonomic treatment was followed by Kirti and Sodhi (2001) and Rose (2002). However, members of the genus Nagiella have been placed in various genera, namely Pleuroptya Meyrick, 1890, Syllepte Hübner, 1823, Patania Moore, 1888 (Inoue 1982; Wang, 1980; Li et al. 2009; Xu 2015; Irungbam et al. 2016; Kirti et al. 2016). Leraut (1997) also listed Nagiella as a junior synonym of Pleuroptya. Kirti and Gill (2007) synonymized Pleuroptya Meyrick, 1890 under Patania on the basis of shared characters such as the lack of gnathos, the valvae leaf-like and without setae, and the presence of distinct cornuti present in the phallus. In Nagiella the gnathos is present, the valvae are broader and bear stout subapical setae, and the cornuti are absent. Based on this morphological evidence and online Lepindex (Beccaloni et al. 2003), we consider that Nagiella warrants distinct generic status and we re-instate it as valid.


The authors are sincerely thankful to Dr. Jean-François Landry, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada for assistance with genitalia description and comments on the manuscript. Special thanks are extended to Dr. Christopher H. Dietrich, University of Illinois at Urbana Champaign, USA and Dr. Murray Fletcher, Orange Agricultural Institute, Orange NSW Australia, for reviewing and improving the manuscript. This study was supported by the National Natural Science Foundation of China (31201733), Natural Science Foundation of Shaanxi Province (2016JM3026), The Chinese Universities Scientific Fund (2452015012), The National Key Research and Development Program (2016YFC0501502) and The Ministry of Science and Technology of the People’s Republic of China (2005DKA21402, 2011FY120200).


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^Misbah Ullah and Zhaofu Yang contribute equally