A new species of the genus Blaptogonia from the Himalayas with four DNA markers (Coleoptera, Tenebrionidae, Blaptini)

Abstract A new species of the genus Blaptogonia Medvedev, 1998, B. zhentanga sp. n., is described from the southern Himalayas of China. Two fragments of mitochondrial protein-coding genes (COI, Cytb), one fragment of mitochondrial ribosomal RNA gene (16S), and one fragment of nuclear rRNA gene (28SD2) of the new species were obtained. A key to the known species of the genus is presented.


Introduction
The tenebrionid genus Blaptogonia Medvedev, 1998 belongs to the subtribe Gnaptorinina Medvedev, 2001 of tribe Blaptini Leach, 1815 within the subfamily Tenebrioninae Latreille, 1802. To date, only four species have been described worldwide and are known to occur only in the southern Himalayas at 3000-4000 meters (Medvedev 2004).
The first species, Trigonoides costulata Fairmaire, 1901, was described from Sikkim, India. It is evident from the text that Trigonoides used by Fairmaire (1901) is an incorrect spelling for Tagonoides Fairmaire, 1886. The second species, Blaps subcarinata Blair, 1927, was described based on the specimens of the third expedition to Mt. Everest from Tibet, China. Subsequently, a new genus, Blaptyscelis, was established by Koch (in Pierre, 1961), along with the combinations Blaptyscelis costulata Fairmaire, 1901and B. subcarinata Blair, 1927. Koch (1965 redefined the diagnostic characters of this genus, which were accepted by Medvedev (1998Medvedev ( , 2004. The third species, Blaptyscelis zurstrasseni Kaszab, 1977 was described from Nepal. A contribution to the knowledge of the tribe Blaptini was subsequently made by Medvedev (1998) who established the replacement name Blaptogonia since Blaptyscelis Koch was originally proposed without a type species designation. Thus, the combinations Blaptogonia costulata Fairmaire, 1901, B. subcarinata Blair, 1927, and B. zurstrasseni Kaszab, 1977were established, and Tagonoides costulata Fairmaire, 1901 was designated as the type species.
At the beginning of the 21st century, Blaptogonia yini Ren, Wang & Yu, 2000 was described from Tibet and placed in Blaptogonia because of the distinct elytral carinae, one of the typical characters of the genus, but was moved recently to the genus Blaps Fabricius, 1775 (Ren et al. 2016) on the basis of additional materials and structures of male genitalia. The fourth species, Blaptogonia tshernjachovskii Medvedev, 2004, was described from Nepal based on two female specimens. Medvedev (2004) also characterized the structures of the apical part of the male abdomen, as well as the ovipositor and genital tubes of the female, as diagnostic characters of the genus Blaptogonia.
In the present study, a new species of the tribe Blaptini, collected from southern Qomolangma Nature Reserve of Tibet, is described. In addition, two fragments of mitochondrial protein-coding genes (COI, Cytb), one fragment of mitochondrial ribosomal RNA gene (16S), and one fragment of nuclear rRNA gene (28SD2) of the new species were sequenced and uploaded to GenBank.

Morphology
All specimens examined in this study were deposited in the Museum of Hebei University (MHBU), Baoding, China. Photographs of morphological structures were taken using a Leica M205A stereomicroscope with a Leica DFC550 camera and Leica application suite 4.6. The habitus photos were taken using a Canon EOS 5D Mark III camera connected to a Canon Macro lens MP-E 65 mm.

DNA extraction, PCR amplification, and sequencing
Total DNA was extracted from leg muscle tissue of a single adult specimen using EZNA® Insect DNA Kit (Omega Bio-tek, USA). Total DNA extract was stored at -20 °C. Two fragments of mitochondrial protein-coding genes (COI, Cytb), one fragment of mitochondrial ribosomal RNA gene (16S), and one fragment of nuclear rRNA gene (28SD2) were amplified using the primers of Table 1. The profile of the PCR amplification consisted of an initial denaturation step at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 50-58 °C for 1 min, and extension at 72 °C for 1 min, and a final 8 min extension step at 72 °C. The PCR products were subsequently checked by 1% agarose gel electrophoresis and sequencing was performed at GENEWIZ Biotech Co., Ltd. (Suzhou, China) using the same primers as in the PCR.  surface with fine granules and irregular and shallow fine punctures, whereas subcarinata has no granules and rows of punctures between carinae and subcarinae; (2) elytral carinae more elevated than the subcarinae, whereas the subcarinae are as high as the carinae in subcarinata; (3) parameres arcuately concave and narrowing from basal 1/5 to apex, whereas the parameres are nearly straight in subcarinata and narrowing from base to apex.

Key to known species of the genus Blaptogonia
Etymology. Named after the type locality, Zhêntang. Description. Head, palps, antennae, carinae, subcarinae, humeral carina, abdomen, tibiae, and tarsus black. Pronotum, elytra, femora, apical spurs, and claws reddish brown to brown. Shiny dorsally and ventrally, with sparse and short pubescence at apex on elytra.
Male (Figs 2a-g, 3). Labrum with sparse punctures and pale yellow setae. Anterior margin of clypeus straight, tilted at sides, surface with sparse fine punctures; frontoclypeal suture shallow. Anterior gena slightly extended before eyes, outer margins straight and converging toward base of clypeus, surface with moderately dense fine punctures; emargination of outer margins of head above antennal base widely obtuse-angular. Eyes transverse, weakly projecting and clearly wider than anterior gena. Posterior gena densely hairy, outer margins arcuately converging to neck. Surface of head with moderately dense punctures. Antennae (Fig. 1b) moderately long, with apical segment reaching beyond pronotal base, length (width) ratio of antennomeres 2 to 11 are 1.0(0. Pronotum (Fig. 2a) transverse, widest before middle, 1.3-1.5 times as wide as long, 1.6-1.8 times as wide as head. Ratio of pronotal width at anterior margin to its maximum and base 0.6: 1.0: 0.9. Anterior margin arcuate, bordered laterally by bead along entire length; posterior margin weakly arcuate, without bead. Anterior angles obtuse, rounded apically; posterior angles obtuse. Pronotal surface between lateral margins weakly convex, with moderately dense and shallow fine punctures on disc. Propleura with fine shallow wrinkles. Prosternal process with dense pale hairs.
Elytra elongate-oval, 1.4-1.6 times as long as wide, and 1.2-1.4 times as wide as pronotum. Each elytron between suture and humeral carina with two distinct carinae. In addition, surface of elytra between suture and 1 st carina, 1 st and 2 nd carina, 2 nd and humeral carina with lower subcarinae; subcarinae between 2 nd and humeral carina indistinct. The humeral carina, subcarinae and carinae reaching base of elytra; 1 st carina fused with humeral carina at apex, 2 nd carina and subcarinae indistinct at apex. Surface of suture, subcarinae, carinae, humeral carina, between suture and subcarinae, between subcarinae and carinae, between subcarinae and humeral carina with irregular sparse and shallow fine punctures, sparse fine granules, and shallow wrinkles; punctures and wrinkles indistinct at apex. Epipleura not reaching suture of elytral angle, outer margin visible in dorsal view only at humeri, sometimes at apices. Visible abdominal sternites covered with short pale recumbent setae, sparse and fine shallow punctures, and fine granules; 1 st to 3 rd ventrites with fine longitudinal wrinkles, 2 nd ventrite flattened in the middle, 4 th ventrite shallowly depressed at sides.
Female 4). Body wider than in male. Antennae reaching pronotal base. Pronotum 1.4-1.5 times as wide as long, 1.6-1.8 times as wide as head. Elytra 1.3-1.5 times as long as wide, and 1.4-1.6 times as wide as pronotum. Second visible sternite not flattened in middle. Apical spur slender, sharp at apex; inner apical spur of protibia slightly longer than outer one. Ventral surface of tarsus without hairy tuft. Spiculum ventrale as in Fig. 1h. Ovipositor as in Fig. 1i.

MG946798
COI AAAAACATGTCTTTTTGTTTTATGATTTAAAGTCTGGCCTGCCCAAT-GATTAATTTTAAATGGCTGCAGTATTTTGACTGTACAAAGGTAG-CATAATCATTAGTTTCTTAATTAGAAGCTGGAATGAATGGTTTGAT-GAAAAATTTACTGTCTCAATTCAATTGTTTTAGAATTTTATTTTTAAGT-GAAAAAGCTTAAATTTTTTAGAAAGACGAGAAGACCCTATAGAGTTTTA-TATGTTTTTTATTATTTATTATATGGTTATAATATTTTTAATTTTAAAATT-TATTTTGTTGGGGTGATGTGAAAATTTAAATAACTTTTCTTAATTT-TAACACTAATTAGTGATTAAATGATCCTTTTTAGGATTAAAAGATTAAAT-TACCTTAGGGATAACAGCGTAATTTTTTTTGAAAGTTCTTATTGA-TAAAAAAGTTTGCGACCTCGATGTTGGATTAAAATTTATTTTTGGTG-TAGAAGCTGGAAAATTTGGGTCTGTTCGACCCTTAAAATTTTACAT-GATCTG Biology. Adults of the new species were found beneath stones in the shrubbery (Fig. 1), usually with more than ten individuals per stone. When threatened, they released quite foul smells and irritating liquids from their abdominal defensive glands. The smell persisted for a few days in the laboratory.