2urn:lsid:arphahub.com:pub:45048D35-BB1D-5CE8-9668-537E44BD4C7Eurn:lsid:zoobank.org:pub:91BD42D4-90F1-4B45-9350-EEF175B1727AZooKeysZK1313-29891313-2970Pensoft Publishers10.3897/zookeys.679.1196011960Research ArticleAnimaliaCrambidaeLepidopteraAgriculture and ForestryAsiaChinaA new cryptic species of Nagiella Munroe from China revealed by DNA barcodes and morphological evidence (Lepidoptera, Crambidae, Spilomelinae)UllahMisbah1misbah2013@nwsuaf.edu.cnYangZhaofu1QiaoPingping1ZhangYalin1https://orcid.org/0000-0002-1204-9181Key laboratory of Plant Protection Resources and Pest Management, Ministry of Education; Entomological Museum, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China
2017080620176796576FF95FFC1-FF8A-FFDE-DD70-FFD7FFC7832E736B7C46-F852-447F-8FCA-90E708B992FE8163762701201705052017Misbah Ullah, Zhaofu Yang, Pingping Qiao, Yalin ZhangThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://zoobank.org/736B7C46-F852-447F-8FCA-90E708B992FE
Nagiellaoccultalis 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 genegenitaliaScopulaquadrimaculalistaxonomyCitation
Ullah M, Yang Z, Qiao P, Zhang Y (2017) A new cryptic species of Nagiella Munroe from China revealed by DNA barcodes and morphological evidence (Lepidoptera, Crambidae, Spilomelinae). ZooKeys 679: 65–76. https://doi.org/10.3897/zookeys.679.11960
Introduction
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 Nagiadesmialis 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: Nagiellainferior (Hampson, 1898), Nagiellaquadrimacualis (Kollar, 1844) with two junior subjective synonyms, desmialis Walker, 1866 and incomitata Swinhoe, 1894, and Nagiellahortulatoides 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 methodsTaxon 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.
Nagiellaoccultalis sp. n.
Nagiellaquadrimaculalis
Nagiellainferior
Pataniaruralis (outgroup)
Nagiellaoccultalis sp. n.
0.0000000
0.0072358
0.0086344
Nagiellaquadrimaculalis
0.0320975
0.000787822
0.0101216
Nagiellainferior
0.0475427
0.0598071
0.000761036
Pataniaruralis (Outgroup)
0.1156349
0.1165689
0.1134248
0.009202714
The diagonal row of values (in bold) indicates intra specific distances, the values below the diagonal indicates mean interspecific distances and values above the diagonal indicates SE estimates obtained by bootstrap procedure (1000 replicates) as implemented in MEGA 6.0. The three species were defined using the 2.0% divergence.
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 Nagiellainferior and selected Pataniaruralis (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.
ResultsDNA 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.
The specific epithet refers to “cryptic”, as this previously undetected species stood within the N.quadrimaculalis complex.
Diagnosis.
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.
Description
(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
Distribution.
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.
Remarks.
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.
Neighbor-joining tree (K2P) based on the 22 COI sequences of the three Nagiella species from China, rooted with Pataniaruralis as outgroup. Bootstrap values <75 are not shown.
Male genitalia A, BN.occultalis sp. n., genitalia slide NAFU PYR 002397 C, DN.quadrimaculalis, genitalia slide NAFU PYR 002069.
https://binary.pensoft.net/fig/139517Discussion
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.
Acknowledgements
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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