Ecologica Montenegrina 18: 18-25 (2018) This journal is available online at: www.biotaxa.org/em

https://zoobank.org/urn:lsid:zoobank.org:pub:93726499-BC6D-4903-AD49-63C42B5C15A1

Barsine vinhphucensis sp. nov. from Vietnam (: : : Lithosiini)

VITALY M. SPITSYN1,2*, ALEXANDER V. KONDAKOV1,2, ALENA A. TOMILOVA1, NHI THI PHAM3,4 & IVAN N. BOLOTOV1,2

1Federal Center for Integrated Arctic Research, Russian Academy of Sciences, 163000, Arkhangelsk, Russia 2Northern Arctic Federal University, 163002, Arkhangelsk, Russia 3Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam 4Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam *Corresponding author: [email protected]

Received 4 May 2018 │ Accepted by V. Pešić: 25 June 2018 │ Published online 29 June 2018.

Abstract 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 by a characteristic pattern and male genitalia structure. It appears to be a local endemic species of the Tam Dao Mountain Range.

Key words: Oriental Region, lichen , Me Linh Biodiversity Station, Tam Dao Mountains.

Introduction

Barsine Walker, 1854 (Erebidae, Arctiinae, Lithosiini) is a diverse and widespread Oriental genus of the lichen moths (Holloway, 2001). The Malay Peninsula, mainland Indochina and the Greater Sunda Islands appear to be the primary evolutionary hotspots harboring the most species-rich Barsine faunas (Holloway, 2001; Černý and Pinratana, 2009; Bucsek, 2012; Volynkin and Černý, 2017a, b, c, 2018), whereas only a few species are distributed on the Moluccas and Lesser Sundas (Holloway, 2001; Spitsyn and Bolotov, 2018). The fauna of this genus in Vietnam is still poorly known. 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

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(Canon, Tokyo, ). 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 MgCl2), 0.8 units Taq DNA polymerase (SibEnzyme Ltd., Russia), and H2O 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).

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

BOLD IDS or Species Collecting locality Sample code GenBank acc. Reference no. Barsine vinhphucensis Vietnam, Tam Dao SPH698 MH345968 This study sp. nov. Mountain Range, Me Linh Biodiversity Station B. vinhphucensis sp. Vietnam, Tam Dao SPH699 MH345969 This study nov. Mountain Range, Me Linh Biodiversity Station B. cuneonotatus , Kanchanaburi SPH686 MH345967 This study (Walker, 1855) Province B. cuneonotatus , , ARB00024464 QMA2182-13 BOLD IDS (Walker, 1855) Xishuangbanna B. lineatus (Walker, Malaysia, Pahang BIOUG06552-E12 LEPMY061-13 BOLD IDS 1855) B. lineatus (Walker, Malaysia, Pahang BIOUG08681-F06 LEPMY541-13 BOLD IDS 1855) B. lineatus (Walker, Malaysia, Pahang BIOUG06552-F01 LEPMY062-13 BOLD IDS 1855) B. pallinflexa Malaysia, Pahang BIOUG06552-F02 LEPMY063-13 BOLD IDS Holloway, 2001 B. senara Moore, Malaysia, Pahang BIOUG14383- LEPMY1136- BOLD IDS [1860] G03 14 B. exclusa Butler, 1877 Malaysia, Pahang BIOUG06552-F08 LEPMY069-13 BOLD IDS

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BARSINE VINHPHUCENSIS SP. NOV. FROM VIETNAM

TABLE 1.

B. exclusa Butler, 1877 Malaysia, Pahang BIOUG08681- LEPMY522-13 BOLD IDS D11 B. punicea Moore, China, Yunnan, ARB00029256 QMA6974-13 BOLD IDS 1878 Xishuangbanna B. perpusilla (Walker, Malaysia, Pahang BIOUG08651-F04 LEPMY444-13 BOLD IDS 1862) B. syntypica Swinhoe, Malaysia, Pahang BIOUG08681-F01 LEPMY536-13 BOLD IDS 1906 lineage 1 B. syntypica Swinhoe, Malaysia, Pahang BIOUG09047- LEPMY573-13 BOLD IDS 1906 lineage 2 A03 B. euprepioides China, Yunnan, ARB00024561 QMA2279-13 BOLD IDS (Walker, 1862) Xishuangbanna B. pulchra (Butler, AYK-04-0841-01 GU696181 GenBank 1877) B. cornicornutata Malaysia, Pahang BIOUG09048- LEPMY747-13 BOLD IDS (Holloway, 1982) G10 B. sp. China, Beijing, Labagoumen LBGM110730.141 KR183263 GenBank B. striata (Bremer & China, Beijing, Dongling DLS100714093 KJ183390 GenBank Grey, 1852) Mountain B. rubricostata Malaysia, Pahang BIOUG14383- LEPMY1139- BOLD IDS (Herrich-Schäffer, G06 14 [1855]) adversata N/A NSGJZ008 KF533485 GenBank (Schaller, 1788)* Tyria jacobaeae N/A DNA1252 KF533506 GenBank (Linnaeus, 1758)* Euplagia N/A NSGJZ003 KF533468 GenBank quadripunctaria (Linnaeus, 1758)* contigua N/A JMZI003 KF533470 GenBank (Walker, 1855)* Brunia dorsalis N/A NSGJZ015 KF533449 GenBank (Walker, 1866)*

*Outgroup taxa.

Description of the species

Barsine vinhphucensis sp. nov. Figs 1-4

Type material. Holotype male, VIETNAM: Vinh Phuc Province, Phuc Yen District, Ngoc Thanh Commune, Tam Dao Mountain Range, Me Linh Station, mountain tropical forest, 21°23.050’N, 105°42.740’E, 370 m alt., 1–13 April 2016, Spitsyn leg. (RMBH, voucher no. Sph0698). Paratype male, same locality and label data (RMBH, voucher no. Sph0699).

Diagnosis. The new species resembles B. punicea melanandra Černý & Pinratana, 2009 by narrow, elongated wings and black coloration of the hindwing (see Černý and Pinratana, 2009: fig. 137a). However,

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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).

Figure 1. Holotype male of Barsine vinhphucensis sp. nov., Tam Dao Mountain Range, Vinh Phuc Province, Vietnam: A) upperside; B) underside. Scale bar = 5 mm. Photos: Vitaly M. Spitsyn.

Description. Male morphology: Wingspan 29 mm, forewing length 14 mm (N = 2). Labial palpus straight and short (slightly longer than eye diameter), brick red. Frons and vertex dark red, vertex in some specimens with brown spot. Antennae fasciculate, long. Thorax dark red. Patagium and tegula brick-red with black spot in the middle. Legs pale red. Abdomen dorsally black, ventrally orange suffused with black. Upperside of forewing dark red with strongly developed black pattern: an irregular black spot in basal area, concave antemedial and straight median wide black lines joined at middle in shape of broad 'X' figure, concave black postmedial line, black lines along veins in outer part of the wing separated by clear dark red lines, black narrow outer margin. Hindwing black, with large dark yellow patch near costal margin. Fringes of fore- and hindwing black. Underside of forewing with reddish-orange proximal half and black distal half. Underside of hindwing black, with large dark yellow patch near costal margin. Male genitalia: Uncus very long, narrow, 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,

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BARSINE VINHPHUCENSIS SP. NOV. FROM VIETNAM 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.

Figure 2. Holotype male genitalia structure of Barsine vinhphucensis sp. nov., Tam Dao Mountain Range, Vinh Phuc Province, Vietnam: A) clasping apparatus, ventral view: v – vinculum, sc – saccus, j – juxta, t – tegumen, u – uncus, bsp – basal saccular process, dsp – distal saccular process, dls – dorsal lobe of distal saccular process, dce – distal costal extension, mcp - medial costal process of valva, alv – apical lobe of valva; B) aedeagus (ae) with vesica everted, lateral left view: bd – basal diverticulum, md1, md2, md3, md4, and md5 – 1st, 2nd, 3rd, 4th, and 5th medial diverticulum, respectively. Scale bar = 1 mm. Photos: Vitaly M. Spitsyn.

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).

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Figure 3. Type locality of Barsine vinhphucensis sp. nov.: mountain tropical forest, Me Linh Biodiversity Station, Tam Dao Mountain Range, Vinh Phuc Province, Vietnam. Photo: Vitaly M. Spitsyn.

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 and Černý 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 and Černý 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.

Acknowledgements

We are very grateful to Dr. Rob De Vos and an anonymous reviewer for their valuable comments on the manuscript. This study was supported by the Russian Ministry of Education and Science (project no. 6.2343.2017/4.6) and Federal Agency for Scientific Organizations (project no. 0409-2015-0143). The project was performed under the framework of an agreement on scientific cooperation between the Institute of Ecology and Biological Resources of VAST (Vietnam) and the Institute of Ecological Problems of the North of the Ural Branch of RAS (Russia). We are grateful to staff of the Me Linh Biodiversity Station for great help during this study.

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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.

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