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DNA barcode reveals the occurrence of Palearctic Olepa schleini Witt et al., 2005 (Lepidoptera: Erebidae: Arctiinae) from peninsular India with morphological variations and a new subspecies

Aparna Sureshchandra Kalawate, Shital Pawara, A. Shabnam & K.P. Dinesh

26 June 2020 | Vol. 12 | No. 9 | Pages: 16143–16152 DOI: 10.11609/jot.5596.12.9.16143-16152

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Journal of Threatened Taxa | www.threatenedtaxa.org | 26 June 2020 | 12(9): 16143–16152 ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print) PLATINUM OPEN ACCESS DOI: htps://doi.org/10.11609/jot.5596.12.9.16143-16152

#5596 | Received 13 December 2019 | Final received 19 May 2020 | Finally accepted 30 May 2020

S h o DNA barcode reveals the occurrence of Palearctc r t Olepa schleini Wit et al., 2005 (Lepidoptera: Erebidae: Arctinae) C o from peninsular India with morphological variatons and a new subspecies m m Aparna Sureshchandra Kalawate 1 , Shital Pawara 2 , A. Shabnam 3 & K.P. Dinesh 4 u n i 1,3,4 Zoological Survey of India, Western Regional Centre, Vidya Nagar, Sector-29, P.C.N.T. (PO), Rawet Road, Akurdi, Pune, c Maharashtra 411044, India. a 2 S.G. Patl Arts, Science & Commerce College, Sakri, Maharashtra 424304, India. t 1 2 3 i [email protected] (corresponding author), [email protected], [email protected], o 4 [email protected] n

Abstract: The present study was taken up to report a new record of Abbreviatons: tl—total length | mw—maximum width | vl—length the tger moth genus, Olepa Watson, 1980 from India along with the of vinculum | vw—maximum width of vinculum | jl—length of juxta discovery of a new subspecies. Earlier the genus was thought to | jw—maximum width of juxta | al—length of aedagus (excluding have restricted distributon range in South and South-East Asia untl vesica) | aw—maximum width of aedagus. the report of O. schleini Wit, Müller, Kravchenko, Miller, Hausmann & Speidel from the Mediterranean Coastal Plain of Israel in 2005. The species identfcaton and the new subspecies is proposed based on the combinaton of morphological studies, available literature The genus Olepa Watson, 1980 was previously comparisons, geographical distributon, DNA barcoding and its phylogeny. Morphological character cryptcity and genital structure considered monotypic, with a single species, O. ricini variatons are well documented in the genus with ‘bio-species’ groups. (Fabricius, 1775). In 1986, Orhant revised the taxonomy DNA Barcoding data of mt COI has provided some resoluton in sortng and systematcs of the genus into two morphological the problems of ‘bio-species’ groups of the genus in the past studies. In the present study, with the available mt DNA COI barcodes and species-groups of O. ocellifera (Walker, 1855) and O. newly generated barcodes genetc identty is confrmed for the species clavatus (Swinhoe, 1885) and he described four new O. ricini, O. schleini, O. toulgoet and Olepa schleini chandrai ssp. species, namely, O. duboisi Orhant, 1986, O. anomi nov., with their phylogenetc relatonships. Morphological variatons within the O. schleini species complex are discussed with a new record Orhant, 1986, O. koslandana Orhant, 1986, and O. of the species for India and a new subspecies descripton. With the toulgoet Orhant, 1986 from southern India and Sri frst mt COI barcode phylogeny for the genus, comments are made on the taxonomic identty of the mt COI DNA barcodes available in the Lanka. Subsequently, four more species were added to GenBank for the Olepa species from India. the genus, thereby making the total 11 from the World Keywords: mt COI gene, Maharashtra, new record, Olepa schleini (see Orhant 2000, 2012; Wit et al. 2005; Dubatolov chandrai ssp. nov., Oriental region, tger moth, Western Ghats. 2011). The caterpillars of this genus feed on various

ZooBank: urn:lsid:zoobank.org:pub:DC0B6F86-AE6F-4115-A71E-5266AC5842A5

Editor: Mandar Paingankar, Government Science College, Gadchiroli, India. Date of publicaton: 26 June 2020 (online & print)

Citaton: Kalawate, A.S., S. Pawara, A. Shabnam & K.P. Dinesh (2020). DNA barcode reveals the occurrence of Palearctc Olepa schleini Wit et al., 2005 (Lepidop- tera: Erebidae: Arctinae) from peninsular India with morphological variatons and a new subspecies. Journal of Threatened Taxa 12(9): 16143–16152. htps://doi. org/10.11609/jot.5596.12.9.16143-16152

Copyright: © Kalawate et al. 2020. Creatve Commons Atributon 4.0 Internatonal License. JoTT allows unrestricted use, reproducton, and distributon of this artcle in any medium by providing adequate credit to the author(s) and the source of publicaton.

Funding: The work is based on the annual research programme of Zoological Survey of India, WRC, Pune ( Ministry of Environment & Forests, Govt. of India).

Competng interests: The authors declare no competng interests.

Acknowledgements: Authors are thankful to Dr. Kailash Chandra, director, Zoological Survey of India, Kolkata and Dr. P.S. Bhatnagar, scientst-D & ofcer-in-charge, Zoological Survey of India, Western Regional Centre, Pune for constant encouragement and research facilites. The frst author wishes to thank Dr. Rahul Joshi (Zoological Survey of India, Gangetc Plains Regional Centre, Patna) for providing literature. Special thanks to Dr. Sameer Kumar Pat, Zoological Survey of India, Western Regional Centre, Pune for tmely help, and for constant encouragement. Authors acknowledge the following survey team members of Zoological Survey of India, Western Regional Centre, Pune for collecton eforts: Dr. V.D. Hegde, Dr. D. Vasanth Kumar, Mr. Namdev R. Gabhale, and Mr. Rakesh Kumar. Thanks are also to Ms. Meharunisa Raje for her help in the wet lab studies. The authors are grateful to the reviewer’s and the subject editor for their valuable suggestons and constructve critcism that improved the manuscript.

16143 J TT DNA barcode reveals the occurrence of Paleartc Olepa schleini Kalawate et al.

crops such as coton, castor, sunfower, sesame, maize, Barcode studies. The outcome of the studies is presented ivy gourd, brinjal, sweet potato, and banana (ICAR- here as a new report of O. schleini from peninsular India NBAIR 2019). with the proposal of a new subspecies of Olepa. Orhant (2000) divided Olepa into two morphological In the past studies, the intraspecies variability among species group based on the shape of uncus and valvae, the species of Olepa is well documented by Wit et al. viz.: the ricini group (uncus and valvae narrow, pointed at (2005); in his words “Within the two groups, the species tp mostly) and ocellifera group (uncus broad, triangular; are slightly variable in habitus and genitalia so that some valvae digitform, apex rounded). He added O. ricini, O. species are doubtul, especially in the ocellifera group”. clavatus, and O. koslandana to the ricini species group Also, O. toulgoet which was synonymised by Singh and O. ocellifera, O. duboisi, O. anomi, and O. kakati & Singh (2013) under O. clavatus is reinstated herein Orhant, 2000 to the ocellifera group. This was further based on distnct features of their genitalia and DNA modifed by Wit et al. (2005) who added O. toulgoet to study (Wit et al. 2005). the ricini species group. The frst set of DNA barcodes for the genus Olepa was provided for the species O. ricini, O. Material and Methods toulgoet, and O. schleini by Wit et al. (2005) during the The moth specimens were collected by using a light new species descripton of O. schleini from Israel. trap during night, and were euthanized by ethyl acetate During our studies in the peninsular India, we found vapors and dry preserved in fumigated entomological some of our Olepa specimens were matching the boxes for further study. The specimens were studied morphological descriptons and male genital characters under Leica EZ4E stereomicroscope. The series of images with the species of O. schleini from Israel. Due to the obtained from the stereomicroscope was stacked using contrastng zoogeographical collecton localites from the CombineZP sofware (Hadley 2010). The geographic Oriental and Palearctc regions and their geographical coordinates and alttude were obtained, possibly by a isolaton, we subjected our samples of Olepa for DNA Garmin GPS. The maps of the collecton locality were

Figure 1. Collecton sites of O. schleini: Indian O. schleini new record to India (star), O. schleini chandrai ssp. nov. (oval).

16144 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 June 2020 | 12(9): 16143–16152 J TT DNA barcode reveals the occurrence of Paleartc Olepa schleini Kalawate et al. prepared using QGIS sofware. The details of collecton Final maximum likelihood (ML) tree was generated locality are given under material examined and also using raxmlGUI v1.3 (Silvestro & Michalak 2012). The shown in Figure 1. The identfcaton of the specimen ML tree (Figure 2) was obtained with 1,000 thorough was done with the help of Orhant (1986) and Wit et al. bootstrap replicates under GTR+I model of nucleotde (2005) terminology followed herein is afer Orhant (1986, substtuton using Akaike Informaton Criterion (-lnL = 2012). The genitalia of male and female were studied 1874.65; AIC = 3935.30) in jModel test (Posada 2008) following Robinson (1976), and terminology is afer Klots and the consensus-generated tree was viewed using Fig. (1970), Orhant (1986, 2012), and Pekarsky (2012). The Tree v1.4.0, treatng species of Pygospila as out-group material examined is deposited in the Zoological Survey (Table 1). of India, Western Regional Centre, Pune, Maharashtra, India (ZSI-WRC). Result and Discussions Our mt COI DNA studies confrmed two genetcally DNA extracton, amplifcaton, and sequencing distnct populatons within the O. schleini species Whole genomic DNA was extracted from the dried complex with morphological variatons. Since the mt leg and abdomen part of pinned preserved adults by COI DNA sequences for our collectons of O. schleini modifed Phenol-Chloroform method (Sambrook & populatons from Nandurbar were matching 100% with Russell 2001) and the resultant pellet was eluted in the mt COI DNA sequences of O. schleini from Israel we 50µL of TE bufer pH 8.0, followed by DNA quanttaton are treatng the Olepa specimens from Nandurbar as the using Qubit 2.0 fuorometer by highly sensitve dsDNA new report of O. schleini from peninsular India. Another assay kit. Approximately 578bp nucleotde porton of populaton of O. schleini from Palghar which is 0.6% the mitochondrial COI gene was amplifed using the divergent for mt COI DNA with the O. schleini from Israel primer pair LepCOI F, 5’-ATTCAACCAATCATAAAGATAT-3’ is described here as a new subspecies under O. schleini. and LepCOI R, 5’-TAAACTTCTGGATGTCCAAAAA-3’ The morphological character variatons across the O. (Hebert et al. 2004). PCR reacton was performed in schleini populatons from Israel (Palaearctc regions) and 25µL reacton volume comprising 12.5µL 2X Master Mix peninsular India (Oriental region) are discussed below (Promega) DNAPolymerase, Reacton Bufer (pH 8.5), (see taxonomic details). 400µM of each dNTPand 4mM MgCl2; 10µM of each Geographically, the type locality of O. schleini forward and reverse primers, 1-5µL DNA (20-100ng) and from Israel and the present record of the species from nuclease free water to Q.S. Thermo cycling profle was peninsular India are separated by a minimum aerial as described by Hebert et al. (2003), with modifcatons, distance of 4,000km. This forms the frst report for an one cycle of 1min at 94°C; 10 cycles of 0.5min at 94°C, Olepa species having its distributon range in both the 1.5min at 45°C and 1.5min at 72°C; 30 cycles of 0.5min Oriental and Palearctc regions at 94°C, 1.5min at 51°C and 1min at 72°C and a fnal In the taxonomic account the comparisons were cycle of 1min at 72°C. PCR products were subjected made on the basis of morphological characters, male to electrophoresis in 1% TAE gel stained with ethidium genitalia, aedagus, positon and number of cornut bromide (EtBr) and visualized under UV light via Gelstain as these characters were considered for comparison Gel Documentaton system. The amplifed PCR product by Orhant (1986, 2000, 2012), Wit et al. (2005), and was purifed using Invitrogen’s Pure Link PCR Purifcaton Dubatolov (2011). The VIIIth abdominal sternite is also Kit. Purifed PCR product was sequenced by Sanger’s considered for comparison as per Orhant (1986, 2000, method on ABI 377 (Applied Biosciences) sequencer 2012) and Dubatolov (2011). outsourced through M/S GeneMatrix, LLP, Pune. Taxonomic account Phylogenetc analysis Family Erebidae Leach, [1815] Generated sequences were initally aligned manually Subfamily Arctinae Leach, [1815] using MEGA version X (Tamura et al. 2011). With our Tribe Arctini Leach, [1815] sequences, mt COI DNA sequences for the species for Genus Olepa Watson, 1980 the genus Olepa (21 sequences) were downloaded from Olepa Watson, 1980, The Generic Names of Moths of the GenBank and aligned. Uncorrected pair-wise genetc the World 2: 133. distances (p-distances) were computed in MEGA version Type species: Alope ocellifera (Walker, 1855) [=Olepa X (Tamura et al. 2011) to delineate our COI sequences ocellifera (Walker, 1855)] from the rest of the Olepa sequences from GenBank.

Journal of Threatened Taxa | www.threatenedtaxa.org | 26 June 2020 | 12(9): 16143–16152 16145 J TT DNA barcode reveals the occurrence of Paleartc Olepa schleini Kalawate et al. based on the 578 bp of mitochondrial COI DNA gene sequences DNA gene COI Olepa based on the 578 bp of mitochondrial Figure 2. Maximum Likelihood (ML) tree for the species of for (ML) tree Likelihood 2. Maximum Figure

16146 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 June 2020 | 12(9): 16143–16152 J TT DNA barcode reveals the occurrence of Paleartc Olepa schleini Kalawate et al.

Olepa schleini Wit, Müller, Kravchenko, Miller, Aedagus (Image 1F). The length of aedagus: 4.34mm, Hausmann & Speidel, 2005 width: 0.597mm. Short, thin, moderately sclerotzed, (Image 1A–G) minutely concave in the middle porton; with single, Material examined: ZSI-WRC L-2028, 04.vii.2019, small, apical spine, apex acute; two-third porton of 01 male, Nandurbar (21.363N & 74.241E; 216m), aedagus near apical spine highly sclerotzed, remaining Maharashtra, India, coll. Shital Pawara. area opposite to it is membranous. Vesica membranous Molecular diagnosis: mt COI DNA sequences of ZSI/ near base; subbasal divertculum moderately scobinated, ENT/311 (GenBank accession number MN822126.1) with two small patches of fve, sclerotsed cornut; were homologous with the sequences of O. schleini medial divertculum heavily scobinated, with two short by Wit et al. (2005) without any nucleotde base pair patches of eight to nine small, diverse cornut, situated diferences. at posterior and anterior porton of divertculum; the Descripton: (Image 1A–B). Forewing length distal divertculum, very heavily scobinated, with two 46mm. Antennae brown and bipectnate, branches patches of cornut, situated medially and terminally, very small. Head and thorax dark grey-brown; collar the medial patch consists of very minute 5–6 cornut bordered with yellow band, with two yellow ringed and the terminal, consists of comparatvely big patch of black spots; patagia with two pairs of big, same sized, small cornut; a unique feature seen, is small patch of yellow ringed black spots; one same bigger spot on moderate sclerotsaton present on the porton between middle of thorax. Abdomen coral, with eight dorsal the basal and the medial divertculum on the ventral and lateral black spots or bands; extremity of abdomen side. yellow; underside of abdomen yellowish. Legs fuscous VIIIth Abdominal sternite: the central plate of brown, except extremity of femura and joints of tarsus VIIIthAbdominal sternite is tridentate, with a very distnct yellowish. Forewing brown, with pale ringed dark spots, patern (Image 1G). incomplete basal line; subbasal line very broad, curved, Measurement (in mm). tl: 5.82, mw: 4.64; vl: 0.772, complete; median, subterminal and terminal lines vw: 1.94; jl: 1.89, jw: 1.56; al: 4.34, aw: 0.597. macular, with variable spots; cilia greyish in between Morphological variatons within O. schleini: The yellowish; underside same patern. Hindwing light coral, forewing of adult of Indian O. schleini is with pale somewhat yellowish near the coastal area, with three ringed macular bands (vs. without pale ringed spots on brown lines or bands; antemedial band thick, complete; forewing of Israelian O. schleini). The subbasal band of median band incomplete; postmedial band with fve forewing is broadest among all members of the group blotches, interrupted in middle; underside same patern (it is normal, not very broad in Israelian O. schleini). as dorsal; cilia greyish in between yellowish. The uncus of Indian O. schleini is narrow and parallel Male Genitalia (Image 1 C–E). Total length of genitalia sided (which is very narrow at the base in Israelian O. 5.82mm, maximum width 4.64mm. Genitalia heavily schleini). The vinculum is broad U-shaped in Indian O. sclerotzed. Uncus oblong, narrow than the other schleini which is of various shapes in Israelian O. schleini members, parallel sided (unlike Israelian O. schleini, (see Wit et al. 2005) but not broad U-shaped. Juxta also where the base of uncus very narrow); apex tapering, difers signifcantly and is very broad in Indian O. schleini. blunt, surrounded by elongated thick setae. Valvae The intraspecies variability is well supported and is in simple, very broad comparatvely, curved in its half consistent with the study of Wit et al. (2005), wherein porton, long, central porton more broad, less narrowed he stated the occurrence of intraspecies variability in and tapering in its distal porton ending in blunt apex; this species and related taxa. The morphometric data in an infecton directed against the uncus; the inner conforms that the genitalial components of O. schleini is margin very broadly serrated at the tp, very distnctly comparatvely smaller than its subspecies. paterned; the outer margin strongly serrated; the tp Distributon: Israel; India (Nandurbar in of the valvae not smooth, directed outward, convex. Maharashtra). Juxta (length: 1.89mm, width: 1.56mm) longer, surai shaped. Vinculum (length: 0.772mm, width: 1.94mm) Olepa schleini chandrai Kalawate, ssp. nov. broad U-shaped, very shorter than tegumen, more than (Image 1H–N) twice wider, uniformly sclerotzed, with small outgrowth urn:lsid:zoobank.org:act:5118E0A1-8041-41DB-9A83-551DD964022E on its proximal end. Tegumen longer than uncus; broad Holotype: ZSI-WRC L-2029, 07.x.2017, 1 male, inverted U-shaped, strongly sclerotsed, comparatvely Suryamal, (19.758N & 73.347E; 518m), Palghar District, smaller. Maharashtra, India, coll. V.D. Hegde and team, GenBank

Journal of Threatened Taxa | www.threatenedtaxa.org | 26 June 2020 | 12(9): 16143–16152 16147 J TT DNA barcode reveals the occurrence of Paleartc Olepa schleini Kalawate et al.

Table 1. GenBank accession numbers for the species of Olepa and Pygospila used in the constructon of Maximum likelihood tree.

GenBank accession number Species Samples from Source

1 MN822127.1 Olepa schleini chandrai Palghar, Maharashtra, India Current studies

2 KY559102.1 Olepa cf. ricini India Unpublished

3 AM050277.1 Olepa schleini Israel Unpublished Nandurbar, Maharashtra, 4 MN822126.1 Olepa schleini Current studies India 5 AM050278.1 Olepa schleini Israel Unpublished

6 AM050279.1 Olepa schleini Israel Unpublished

7 AM050275.1 Olepa schleini Israel Unpublished

8 AM050271.1 Olepa schleini Israel Unpublished

9 AM050270.1 Olepa schleini Israel Unpublished

10 AM050276.1 Olepa schleini Israel Unpublished

11 KX050259.1 Olepa schleini NA Ronka et al. (2016)

12 AM050273.1 Olepa schleini Israel Unpublished

13 AM050272.1 Olepa schleini Israel Unpublished

14 AM050274.1 Olepa schleini Israel Unpublished

15 AM050285.1 Olepa toulgoet India Unpublished

16 AM050286.1 Olepa toulgoet India Unpublished

17 KM985648.1 Olepa cf. ricini Malappuram, Kerala, India Unpublished

18 KY559103.1 Olepa sp. India Unpublished

19 AM050284.1 Olepa ricini India Unpublished

20 AM050281.1 Olepa ricini India Unpublished

21 AM050283.1 Olepa ricini India Unpublished

22 AM050280.1 Olepa ricini India Unpublished

23 AM050282.1 Olepa ricini India Unpublished

24 HQ953032.1 Pygospila hyalotypa Australia Unpublished

25 HQ953030.1 Pygospila hyalotypa Australia Unpublished

26 HQ953031.1 Pygospila hyalotypa Australia Unpublished

27 HQ953029.1 Pygospila bivitalis Australia Unpublished

28 HQ953027.1 Pygospila bivitalis Australia Unpublished

29 JX017862.1 Pygospila bivitalis Australia Haines & Rubinof (2012)

30 HQ953028.1 Pygospila bivitalis Australia Unpublished

31 KF392550.1 Pygospila tyres Australia Hebert et al. (2013)

32 HQ953033.1 Pygospila tyres Australia Unpublished

33 HQ953036.1 Pygospila tyres Australia Unpublished

34 HQ990824.1 Pygospila tyres Pakistan Unpublished

35 HQ990826.1 Pygospila tyres Pakistan Unpublished

36 HQ990825.1 Pygospila tyres Pakistan Unpublished

37 HQ990828.1 Pygospila bivitalis Pakistan Unpublished

38 HQ990827.1 Pygospila tyres Pakistan Unpublished

39 HQ953034.1 Pygospila tyres Australia Unpublished

40 KX862292.1 Pygospila tyres Pakistan Ashfaq et al. (2017)

41 KT988774.1 Pygospila tyres NA Unpublished

42 HQ953035.1 Pygospila tyres Australia Unpublished

43 GU695393.1 Pygospila marginalis Papua New Guinea Unpublished

16148 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 June 2020 | 12(9): 16143–16152 J TT DNA barcode reveals the occurrence of Paleartc Olepa schleini Kalawate et al. accession number MN822127.1. oblong, narrow, tapering towards apex, with blunt apex, Diferental diagnosis: This subspecies could be long, surrounded by elongated, thin, minute setae. distnguished from the other taxa in the genus by a Tegumen shouldered, longer than uncus, broad inverted combinaton of the following characters: thick median U-shaped, moderately sclerotsed. Valvae curved, long, line of hindwing cojoined to antemedial line in some central porton broad, cucullus narrowing and ending places; uncus long with blunt apex; valvae with slender in a blunt apex, in an infecton directed against uncus, apex, more recurved, the infecton directed against costa plough-like; the inner margins broadly serrated in the uncus; juxta broader in its basal part and bigger; the upper region, the outer margin minutely serrated, the central plate of VIIIth Abdominal sternite distnctly tp of valvae not smooth, directed outward, convex. tridentate resembling a frill. The positon of cornut in Juxta (length: 1.72mm, width: 1.66mm) broader in its the new subspecies is almost similar to O. neumuthi basal part and bigger. Vinculum (length: 0.797mm, Orhant, 2012, except cornut in the new subspecies is width: 1.84mm) truncated U-shaped, very shorter than stronger, longer, denser, and present only in the subbasal tegumen, uniformly sclerotzed, two tmes wider, with region of vesica. moderate outgrowth on its proximal end. Molecular diagnosis: Pair wise genetc distance Aedagus (Image 1M). Length: 5.10mm, width: was 0.6% for the mt COI DNA sequences of O. schleini 0.687mm; is closer to O. neumuthi. It is long, thick, chandrai ssp. nov. from peninsular India and O. schleini crooked, with single large, stout, apical spine, the tp from Israel; 1.8% to 2.% from O. toulgoet and 25.4% from of spine rounded; two-third porton of the aedagus O. ricini. Due to limitatons with mt COI DNA sequences near apical spine more sclerotsed than the rest of further genetc studies are warranted to elevate the taxa the aedagus, the ramianing area opposite to it, is to the species rank. membranous, not sclerotsed, the whole aedagus except Descripton: (Image 1 H–I). Forewing length 48mm. the porton mentoned, is minutely sclerotsed; vesica Head and thorax dark grey-brown with admixture of membranous, with patches of scobinaton, armed with olive-green scales; palpi brown; collar bordered with stout, very long cornut in three patches present on the a yellow band and two black spots surrounded with subbasal region, comparatvely dense cornut. whitsh-yellow ring; patagia with two pairs of whitsh- VIIIth Abdominal sternite: the central plate of it is yellow ringed black spots, the front spot smaller; one distnctly tridentate, resembles a frill, present in double same spot, bigger, on middle of thorax. Abdomen coral, layer (Image 1N). with black bands, the frst two are shorter, not reaching Measurement of genitalia (in mm). tl: 6.00, mw: 5.44; the lateral side, others reaching the lateral side of vl: 0.797, vw: 1.84; jl: 1.72, jw: 1.66; al: 5.10, aw: 0.687. abdomen, lateral spots present, extreimity of abdomen Etymology: The subspecies is named afer Dr. Kailash black; underside of abdomen yellowish. Legs fuscous Chandra, an eminent Entomologist and the Director of brown, except extremity of femura and joints of tarsus the Zoological Survey of India, Kolkata. yellowish. Forewing moderately broad, apex rounded, Remarks: The new subspecies is externally similar to fuscous brown, with several black spots surrounded by O. neumuthi and belongs to the ricini-schleini-neumuthi whitsh-yellow ring, these spots sometmes fused to form complex. It difers from all congeners by the distnct band or line; basal and subbasal bands are not complete; cucullus, which is narrowing and ending in a blunt antemedial band thick; median and subterminal bands apex, (vs. cucullus of O. ricini and O. neumuthi always complete; terminal line as a row of blackish dots ending in a point or less rounded apex; in O. schleini, it between the veins; cilia brownish, in between patches is rounded). The infecton of valvae is directed towards of of-white. Hindwing light coral, costal area yellowish, the uncus in O. ricini, O. neumuthi, and O. schleini. with dark brown spots or blotches, these spots Whereas, it is directed against the uncus in the new sometmes unites to form band or line; antemedial band subspecies. The vinculum is truncated U-shaped, very thick, curved; thick median band cojoined to antemedial shorter than the tegumen, with moderate outgrowth on band in some places; postmedial band interrupted in the its proximal end (vs. U-shaped in O. ricini, and O. schleini middle; thin marginal band; underside patern of both with comparatvely large and moderate outgrowth, the wings are exactly same as the dorsal; cilia brownish, respectvely; in O. neumuthi it is V-shaped, with small in between patches of of-white. outgrowth). Male Genitalia (Image 1J–L). The total length of The aedagus of the new subspecies is similar to the genitalia 6.00mm, width 5.44mm. Genitalia less O. neumuthi. Aedagus of O. schleini chandrai ssp. nov. is sclerotsed comparatvely. Uncus fused with tegumen, thick, crooked and strong, with three patches of strong,

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longer and dense cornut in the subbasal region of vesica Suryamal, Palghar District (Western Ghats), Maharashtra, (vs. almost straight aedagus, presence of three patches India. of modest cornut in the basal region in O. neumuthi; in The present study is indicatve of the species richness O. schleini and O. ricini there are 5–6 patches of cornut). of this genus which was underestmated due to its Distributon: Known only from the type locality: complex cryptc nature and wide range of distributon. A

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Table 2. Genetc distance (in percentage) matrix for the mt COI DNA COI DNA gene data are available specifcally only for O. among the DNA Barcode available species of Olepa. toulgoet, O. ricini, O. schleini, and O. schleini chandrai Olepa Olepa Olepa ssp. nov. Voucher specimen studies on the two lineages schleini Olepa ricini schleini toulgoet chandrai of O. cf. ricini (KM985648.1) and Olepa sp. (KY559103.1) Olepa schleini could provide informaton on the mt COI DNA barcode Olepa schleini data for the known species of Olepa or it may be a 0.60 chandrai potental new species awaitng formal descripton. As Olepa 1.60 2.30 toulgoet stated earlier, the new subspecies is proposed on the Olepa ricini 25.90 25.90 25.40 basis of the following set of characters: it possesses a thick median line of hindwing cojoined to antemedial line in some places; uncus long and valvae with slender total of 21 mt COI sequences of Olepa were downloaded apex, more recurved, the infecton directed against from the GenBank, with our two mt COI sequences on the uncus; the central plate of VIIIth abdominal sternite the phylogenetc tree fve clear sub-clades could be distnctly tridentate resembling a frill. The positon discerned within the larger ‘Olepa clade’. Our sequences of cornut in the new subspecies is almost similar to of Olepa species formed a monophyletc clade with the O. neumuthi, except cornut in the new subspecies species of O. schleini, over all genetc distance within is stronger, longer and denser and present only in the the ‘Olepa schleini species clade’ was up to 0.6%. Since subbasal region of vesica. our sequence for O. schleini (Nandurbar) was matching This study forms the frst report for the O. schleini 100% with the 11 Olepa schleini sequences of Wit et described from Palearctc region having its distributon in al. (2005) we have treated our sample of O. schleini Oriental region which is around 4,000km apart invoking from Nandurbar, Maharashtra as the frst record of O. many zoogeographic queston of species distributon schleini to Indian sub-contnent extending its range of spatally. Understanding the genetc heterogeneity and distributon from Israel in spite of slight morphological morphological divergences within the species of Olepa diferences (see taxonomic discussion above). from the studies of Wit et al. (2005) and the current Our Olepa species collected from Palghar, study, there could be many more new species awaitng Maharashtra is showing a very shallow genetc formal descripton which can be justfed through spatal divergence of 0.6% from O. schleini populatons of India sampling and DNA studies. and Israel with high morphological divergence. Hence, O. schleini collectons from Palghar has been proposed as References a subspecies (O. schleini chandrai ssp. nov.) of O. schleini due to high morphological divergence (i.e., hindwing Ashfaq, M., S. Akhtar, M.A. Raf, S. Mansoor & P.D. Hebert (2017). Mapping global biodiversity connectons with DNA barcodes: patern, distnct cucullus and the positon of cornut in Lepidoptera of Pakistan. PLoS ONE 12(3): e0174749. htps://doi. the new subspecies) and shallow genetc distance (Table org/10.1371/journal.pone.0174749. 2) warrantng further molecular studies. Spatally O. Dubatolov, V.V. (2011). A new Olepa Watson, 1980 species from South India (Lepidoptera, Arctidae). Atalanta 42 (1/2) : 136–137. schleini from Nandurbar and O. schleini chandrai ssp. Fabricius, J.C. (1775). Systema Entomologiae, sistens Insectorum nov. from Palghar are 180km apart. Shallow genetc Classes, Ordines, Genera, Species, Adiects Synonymis, Locis, divergence of 1.8% was earlier reported between the Descriptonibus, Observatonibus. Korti, Flensbvrgi et Lipsidae [= Flensburg and Leipzig]: [32]+832pp. species O. ricini and O. toulgoet by Wit et al. (2005). Hadley, A. (2010). Combine ZP sofware, new version. Available from: Interestngly, sequence of O. ricini (KY559102.1) from http://www.hadleyweb.pwp.blueyonder.co.uk/CZP/News.htm India matches 100% with the sequences O. schleini (Figure (accessed 16 August 2018). Haines, W.P. & D. Rubinof (2012). Molecular phylogenetcs of the 2), where voucher specimen studies are warranted to moth genus Omiodes Guenée (Crambidae: Spilomelinae), and understand the morphological divergence. Also, the the origins of the Hawaiian lineage. Molecular Phylogenetcs and sequence of O. ricini (KM985648.1) from Malappuram, Evoluton 65(1): 305–316. Hebert, P.D., A. Cywinska, S.L. Ball & J.R. Dewaard (2003). Biological Kerala do not match either of the sequences of O. ricini identfcatons through DNA barcodes. Proceedings of the Royal or O. schleini, where morphological studies of voucher Society of London. Series B: Biological Sciences 270(1512): 313–321. samples are warranted for the exact identty of the htps://doi.org/10.1098/rspb.2002.2218 Hebert, P.D., E.H. Penton, J.M. Burns, D.H. Janzen & W. Hallwachs species. Likewise, the voucher specimen of the sequence (2004). Ten species in one: DNA barcoding reveals cryptc species in of Olepa sp. (KY559103.1) from India needs examinaton the neotropical skipper buterfy Astraptes fulgerator. Proceedings of the Natonal Academy of Sciences 101(41): 14812–14817. htps:// to justfy the species name for the sequence. As of now doi.org/10.1073/pnas.0406166101. among the 11 species of Olepa available globally mt Hebert, P.D., J.R. Dewaard, E.V. Zakharov, S.W. Prosser, J.E. Sones,

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J.T. McKeown, B. Mantle & J. La Salle (2013). A DNA ‘barcode blitz’: Rönkä, K., J. Mappes, L. Kaila & N. Wahlberg (2016). Putng Parasemia rapid digitzaton and sequencing of a natural history collecton. PLoS in its phylogenetc place: a molecular analysis of the subtribe ONE 8(7): e68535. htps://doi.org/10.1371/journal.pone.0068535 Arctina (Lepidoptera). Systematc Entomology 41(4): 844–853. ICAR-NBAIR (2019). Olepa ricini (Fabricius) Natonal Bureau of Sambrook, J. & D. Russell (2001). Molecular Cloning: A Laboratory Agricultural Insect Resources. Available from: htp://www.nbair.res. Manual, 3rd Editon. 619 Cold Spring Harbor, 2100pp. in/insectpests/credits.php (accessed on 18 September 2019). Singh, N. & J. Singh (2013). Review of the genus Olepa Watson Klots, A.B. (1970). Lepidoptera, pp. 115–130. In: Tuxen, S.L. (ed.). (Lepidoptera: Erebidae: Arctinae). Tinea 22(4): 272–277. Taxonomist’s glossary of genitalia in insects. Munksgaard, Silvestro, D. & I. Michalak (2012). Raxml GUI: A graphical front-end for Copenhagen, 359pp. RAxML. Organisms Diversity & Evoluton 12: 335−337. https://doi. Orhant, G. (1986). Deuxième contributon à l’étude des org/10.1007/s13127-011-0056-0 lépidoptèreshétérocères du sud-estAsiatque (1) Le Swinhoe, C. (1885). Proceedings of the general meetngs for scientfc complexed’espèces “ricini Fabricius” (Arctidae: Arctinae). Bulletn business of the Zoological Society of London, 295pp. Society Science Natonal France 50: 9–22. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei & S. Kumar Orhant, G. (2000). Unnouvel Olepa de l’Inde (Lepidoptera, Arctidae, (2011). MEGA5: Molecular Evolutonary Genetcs Analysis using Arctinae). Lambillionea100 (2): 269–270. Maximum Likelihood, Evolutonary Distance and Maximum Orhant, G. (2012). Olepa neumuthi new species from Thailand Parsimony Methods. Molecular Biology and Evoluton 28: (Lepidoptera: Arctidae). Bulletn Society Entomology Mulhouse 2731−2739. htps://doi.org/10.1093/molbev/msr121 68(4): 61–64. Walker, F. (1855). List of the specimens of Lepidopterous insects in the Pekarsky, O. (2012). Two new species of Pseudohadena Alphéraky, collecton of the Britsh Museum 3: 583−775. 1889 from Kazakhstan (Lepidoptera, Noctuidae, Xyleninae). ZooKeys Wit, T.J., G.C. Müller, V.D. Kravchenko, M.A. Miller, A. Hausmann & 187: 9–34. htps://doi.org/10.3897/zookeys.187.2661 W. Speidel (2005). A new Olepa species from Israel (Lepidoptera, Posada, D. (2008). J Model Test: Phylogenetc Model Averaging. Arctidae). Nachrichtenblat der Bayerischen. Entomologen 54(3/4): Molecular Biology and Evoluton 25: 1253−1256. htps://doi. 101–115. htp://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1 org/10.1093/molbev/msn083 .1.463.9720&rep=rep1&type=pdf Robinson, G.S. (1976). The preparaton of slides of Lepidoptera genitalia with special reference to the Microlepidoptera.

Entomologist’s Gazete 27: 127–132. Threatened Taxa

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PLATINUM The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservaton globally by publishing peer-reviewed artcles online every month at a reasonably rapid rate at www.threatenedtaxa.org. OPEN ACCESS All artcles published in JoTT are registered under Creatve Commons Atributon 4.0 Internatonal License unless otherwise mentoned. JoTT allows allows unrestricted use, reproducton, and distributon of artcles in any medium by providing adequate credit to the author(s) and the source of publicaton.

ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print)

June 2020 | Vol. 12 | No. 9 | Pages: 15967–16194 Date of Publicaton: 26 June 2020 (Online & Print) www.threatenedtaxa.org DOI: 10.11609/jot.2020.12.9.15967-16194

Communicatons DNA barcode reveals the occurrence of Palearctc Olepa schleini Wit et al., 2005 (Lepidoptera: Erebidae: Arctinae) from peninsular India with morphological variatons Dusky Langurs Trachypithecus obscurus (Reid, 1837) (Primates: Cercopithecidae) in and a new subspecies Singapore: potental origin and conficts with natve primate species – Aparna Sureshchandra Kalawate, Shital Pawara, A. Shabnam & K.P. Dinesh, Pp. 16143– – Andie Ang, Sabrina Jabbar & Max Khoo, Pp. 15967–15974 16152

A new report on mixed species associaton between Nilgiri Langurs Semnopithecus johnii Present status of the genus Sphrageidus Maes, 1984 (Lepidoptera: Erebidae: and Tufed Grey Langurs S. priam (Primates: Cercopithecidae) in the Nilgiri Biosphere Lymantriinae) from India Reserve, Western Ghats, India – Amritpal Singh Kaleka, Devinder Singh & Gagan Preet Kour Bali, Pp. 16153–16160 – K.S. Chetan Nag, Pp. 15975–15984 Early stages of Nilgiri Grass Yellow Eurema nilgiriensis (Yata, 1990) (Lepidoptera: A review of the bacular morphology of some Indian bats (Mammalia: Chiroptera) Pieridae), with a note on its range extension in the Kerala part of the Western Ghats, – Bhargavi Srinivasulu, Harpreet Kaur, Tariq Ahmed Shah, Gundena Devender, Asad Gopi, India Sreehari Raman & Chelmala Srinivasulu, Pp. 15985–16005 – Balakrishnan Valappil & V.K. Chandrasekharan, Pp. 16161–16165

Status of the Critcally Endangered Bengal Florican Houbaropsis bengalensis (Gmelin, 1789) in Koshi Tappu Wildlife Reserve, Nepal Notes – Hem Sagar Baral, Tek Raj Bhat, Sailendra Raj Giri, Ashok Kumar Ram, Shyam Kumar Shah, Laxman Prasad Poudyal, Dhiraj Chaudhary, Gitanjali Bhatacharya & Rajan Amin, Breeding site records of three sympatric vultures in a mountainous clif in Kahara- Pp. 16006–16012 Thathri, Jammu & Kashmir, India – Muzafar A. Kichloo, Sudesh Kumar & Neeraj Sharma, Pp. 16166–16169 Observatons on breeding behaviour of a pair of endangered Egyptan Vultures Neophron percnopterus (Linnaeus, 1758) over three breeding seasons in the plains of Punjab, India First distributon record of Elongated Tortoise Indotestudo elongata (Blyth, 1853) – Charn Kumar, Amritpal Singh Kaleka & Sandeep Kaur Thind, Pp. 16013–16020 (Reptlia: Testudines: Testudinidae) from Bihar, India – Arif, Sourabh Verma, Ayesha Mohammad Maslehuddin, Utam, Ambarish Kumar Mall, Additons to the cicada (Insecta: Hemiptera: Cicadidae) fauna of India: frst report and Gaurav Ojha & Hemkant Roy, Pp. 16170–16172 range extension of four species with notes on their natural history from Meghalaya –Vivek Sarkar, Cuckoo Mahapatra, Pratyush P. Mohapatra & Manoj V. Nair, Pp. 16021–16042 The niche of shrimp stocks (Xiphopenaeus kroyeri Heller, 1862) from southeastern Brazil: a stable isotope approach The perceptons of high school students on the habitat of the crab Ucides cordatus – Keltony de Aquino Ferreira, Leandro Rabello Monteiro & Ana Paula Madeira Di (Linnaeus, 1763) (Crustacea: Decapoda: Ucididae) in northern Rio de Janeiro State, Benedito, Pp. 16173–16176 southeastern Brazil – Laiza Fernanda Quintanilha Ribeiro, Laura Helena de Oliveira Côrtes & Ana Paula Madeira First record of the White Tufed Royal Pratapa deva lila Moore, [1884] (Lepidoptera: Di Benedito, Pp. 16043–16047 Lycaenidae: Theclinae) from Himachal Pradesh, extending its known range westwards – Sanjay Sondhi, Pp. 16177–16179 Woody species diversity from proposed ecologically sensitve area of northern Western Ghats: implicatons for biodiversity management Range extension of the Lilac Silverline Apharits lilacinus to southern Rajasthan and a – M. Tadwalkar, A. Joglekar, M. Mhaskar & A. Patwardhan, Pp. 16048–16063 review of the literature –K.S. Gopi Sundar, Swat Kitur, Vijay Kumar Koli & Utkarsh Prajapat, Pp. 16180–16182 Resolving taxonomic problems in the genus Ceropegia L. (Apocynaceae: Asclepiadoideae) with vegetatve micromorphology A record of gynandromorphism in the libellulid dragonfy Crocothemis servilia (Insecta: – Savita Sanjaykumar Rahangdale & Sanjaykumar Ramlal Rahangdale, Pp. 16064–16076 Odonata) from India – R.V. Renjith & A. Vivek Chandran, Pp. 16183–16186 A checklist of angiosperm fora of low elevaton lateritc hills of northern Kerala, India – K.A. Sreejith, V.B. Sreekumar, P. Prashob, S. Nita, M.P. Prejith & M.S. Sanil, Pp. 16077– Carcass consumpton by Nasuttermes callimorphus (Blatodea: Isoptera) in highland 16098 forests from Brazil – Igor Eloi, Mário Herculano de Oliveira & Maria Avany Bezerra-Gusmão, Pp. 16187– Phytodiversity of chasmophytc habitats at Olichuchatam Waterfalls, Kerala, India 16189 – Arun Christy & Binu Thomas, Pp. 16099–16109 New records of nasutform termite (Nasuttermitnae: Termitdae: Isoptera) from Contributon to the macromycetes of West Bengal, India: 51–56 Meghalaya, India – Diptosh Das, Entaj Tarafder, Meghma Bera, Anirban Roy & Krishnendu Acharya, – Khirod Sankar Das & Sudipta Choudhury, Pp. 16190–16192 Pp. 16110–16122

Corrigendum Short Communicatons Correctons to A citzens science approach to monitoring of the Lion Panthera leo Catalogue of herpetological specimens from peninsular India at the Sálim Ali Centre for (Carnivora: Felidae) populaton in Niokolo-Koba Natonal Park, Senegal Ornithology & Natural History (SACON), India – Dimitri Dagorne, Abdoulaye Kanté & John B. Rose, Pp. 16193–16194 – S.R. Ganesh, S. Bhupathy, P. Karthik, G. Babu Rao & S. Babu, Pp. 16123–16135

Osteological descripton of Indian Skipper Frog Euphlycts cyanophlycts (Anura: Dicroglossidae) from the Western Ghats of India – Pankaj A. Gorule, Sachin M. Gosavi, Sanjay S. Kharat & Chandani R. Verma, Pp. 16136– Publisher & Host 16142

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