Barsine vinhphucensis sp . nov . from Vietnam ( Lepidoptera : Erebidae : Arctiinae : Lithosiini )

Barsine vinhphucensis sp. nov. (Lepidoptera: Erebidae: Arctiinae) has been discovered from mountain tropical forest of northern Vietnam. The new species differs from all the other members of the genus by a characteristic pattern and male genitalia structure. It appears to be a local endemic species of the Tam Dao Mountain Range.

The present study describes Barsine vinhphucensis sp.nov.that has been discovered near the Me Linh Biodiversity Station in mountain tropical forest of northern Vietnam.

Material and Methods
This study is based on material from the collection of the Russian Museum of Biodiversity Hotspots (RMBH thereafter) of the Federal Center for Integrated Arctic Research of the Russian Academy of Sciences, Arkhangelsk, Russia.The genitalia were dissected and mounted on a glass slide with Histofluid® (Paul Marienfeld GmbH & Co., Germany).The images of specimens were taken with a Canon EOS 650D camera (Canon, Tokyo, Japan).The photos of the genitalia were obtained using a research stereomicroscope (SteREO Discovery.V8, Carl Zeiss, Germany).
Total DNA was extracted from a single leg of each dry specimen according to standard phenol/chloroform procedures (Sambrook et al., 1989).The barcode region of the mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified and sequenced using primers LCO1490 and HCO 2198 (Folmer et al., 1994).The PCR mix contained approximately 200 ng of total cell DNA, 10 pmol of each primer, 200 μmol of each dNTP, 2.5 μl of PCR buffer (with 20 mmol MgCl 2 ), 0.8 units Taq DNA polymerase (SibEnzyme Ltd., Russia), and H 2 O was added for a final volume of 25 μl.Temperature cycling was as follows: 95°C (5 min), 40 cycles of 95°C (50 sec), 48°C (50 sec), 72°C (50 sec) and a final extension at 72°C (5 min).Forward and reverse sequencing was performed on an automatic sequencer (ABI PRISM® 3730, Applied Biosystems) using an ABI PRISM® BigDye™ Terminator v. 3.1 reagent kit.The resulting sequences were analyzed with BioEdit 7.0.9(Hall 1999).The first base pairs behind the 3` end of each primer were difficult to read, and the sequences were shortened to a 527 bp fragment.
For phylogenetic analyses, 21 available COI haplotypes of Barsine spp.were obtained from NCBI GenBank and BOLD Database (Table 1).Sequences of five other Arctiinae taxa were used as an outgroup.The sequence dataset was aligned using the ClustalW algorithm (Thompson et al. 1994) implemented in MEGA6 (Tamura et al. 2013).Bayesian phylogenetic analyses were performed in MrBayes v. 3.2.6 (Ronquist et al. 2012) as described in Bolotov et al. (2017).The best evolutionary models for each partition based on the corrected Akaike Information Criterion (AICc) of MEGA6 (Tamura et al. 2013) were as follows: (1) 1st codon of the COI: GTR+G (G = 0.45); (2) 2nd codon of the COI: TN93+G (G = 0.05); (3) 3rd codon of the COI: HKY+G (G = 0.05).The resulting tree was reconstructed using Archaeopteryx v. 0.9901 (Han and Zmasek 2009).137a).However, B. vinhphucensis sp.nov.differs from this taxon by the forewing pattern (Fig. 1), i.e. by wide antemedial and median lines (vs narrow), straight median line (vs incurved), lack of a spot in discocellular (vs presence of a black spot), black lines along veins in outer part of the wing separated by clear dark red lines (vs black, suffused with crimson), and much larger wingspan of 29 mm (vs.13-17 mm).The male genitalia of Barsine vinhphucensis sp.nov.(Fig. 2) differs from that of B. punicea melanandra by having a basal process of sacculus longer than the length of valve (vs.shorter than length of valve) and short, rounded saccus (vs elongated, V-shaped).with small sharp claw-like apex (Fig. 2).Tegumen short, V-shaped.Juxta broad, W-shaped with a shallow recess medially, moderately sclerotized.Saccus rounded and short.Valva broad; medial costal process large, elongated, with rounded apex, strongly sclerotized and covered with tiny teeth; distal costal extension small, weakly sclerotized; apical lobe of valva rounded, narrow; sacculus strongly sclerotized, its basal process longer than the length of valva, strongly curved, gradually tapering posteriorly, pointed apically; distal saccular process small, short, tooth-like; dorsal lobe of distal saccular process small, tooth-like.Aedeagus short and broad, straight.Vesica large, broad; basal diverticulum large, sack-like, bilobate, apically with scobination (proximal lobe) and long thin cornuti (distal lobe); 1st medial diverticulum long, sack-like, distally with weak scobination; 2nd medial diverticulum broad, covered by thick short cornuti of different size; 3rd medial diverticulum long, straight, slightly tapering distally, with thick short cornuti of different size; 4th medial diverticulum moderately large, broad, covered by strong short cornuti; 5th medial diverticulum broad, with dense scobination.Female.Not known.

DNA data.
Reference COI sequence nos.MH345968 (holotype) and MH345969 (paratype).With respect to the COI sequences, the new species is closely allied to Barsine pulchra, B. striata, and B. sp.(K2P distance = 4.6-5.9%;see Table 1 for accession numbers of sequences).According to our Bayesian phylogenetic model, Barsine vinhphucensis sp.nov.appears to be a close relative of B. pulchra (Fig. 4), although the latter species is very different externally.Moreover, the male genitalia of B. pulchra differs from those of the new species by much shorter basal saccular process, which is not exceed a half of the valva length, and by the absence of medial costal process.
Etymology.The name of the new species is derived from Vietnam's Vinh Phuc Province, in which the types of the species were collected.

Distribution.
Only known from the type locality in Vietnam, Tam Dao Mountain Range (Fig. 3).

Discussion
Although recent taxonomic works have increased our knowledge on the species richness of Barsine from the poorly studied faunas of Southeast and East Asia (e.g.Černý and Pinratana 2009;Bucsek 2012;Černý and Volynkin 2016;Bayarsaikhan et al. 2017;Volynkin andČerný 2017a, b, c, 2018;Volynkin 2018;Spitsyn and Bolotov 2018), the revision of this large and species-rich genus is very far from being complete.In summary, this genus comprises approximately a hundred of valid species-and subspecies-level taxa, many of which were discovered during the last two decades (Volynkin 2018).Several new species were recently described from Vietnam (Volynkin andČerný 2016, 2017a).
In this paper, we describe an additional Vietnamese species, Barsine vinhphucensis sp.nov., which has been discovered from the Tam Dao Mountain Range.This species remotely resembles B. punicea melanandra but it could be distinguished by the remarkable pattern, larger size, and male genitalia structure.The nominative subspecies of B. punicea is phylogenetically distant from Barsine vinhphucensis sp.nov.(Fig. 4), sequences of B. punicea melanandra are not available.

Figure 4 .
Figure 4. Majority-rule consensus Bayesian phylogenetic tree of Barsine spp.based on available COI sequences.The new species name is in red.Numbers near nodes are Bayesian posterior probabilities.

Table 1 .
List of COI sequences examined in this study.