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First Indian DNA barcode record for the moth species Pygospila tyres ( Cramer , 1780 ) ( Lepidoptera : Crambidae : Spilomelinae ) distributed in Asia and Australia

The species Pygospila tyres was described from the Coromandel region of India about 240 years ago, accommodated in the family Crambidae having immense importance. The species is morphologically cryptic and is known to have 10 extant species under the genus. Earlier mt DNA Barcodes for the species were available from Pakistan, Korea, and Australia, here we report the first barcode of the species from the country of its type locality. Morphological details for the collections with the male and female genitalia are provided for the taxonomic identification. Identities of the mt COI DNA sequences for the genus in the GenBank are discussed.

ISSN 0974-7907 (Online); ISSN 0974-7893 (Print) The members of the superfamily Pyraloidea are known to cause crop yield loss between 10 to 100 per cent across the world (Jotwani & Young 2007). Earlier the family Crambidae was originally a part of the family Pyralidae, but separated from it by Munroe (1972). They are of immense economic importance as they are the pest on many agricultural important cash crops like sugarcane and other crops like maize, brinjal, tomato, cabbage, cotton, oil seed, and bamboo (Solis 1997). Most of the crambid moths are morphologically cryptic (cryptic species is a group of individuals that are morphologically identical to each other but belong to different species) and difficult to study. The moths of the subfamily are characterized by the absence of chaetosemata, presence of bilobed subcostal retinaculum in male, praecinctorium fornix tympani projecting and pointed spinula. Corpus bursae in the female genitalia lack signum and gnathos absent (Minet 1981;Solis & Maes 2003;Solis 2007;Kumar et al. 2013

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added. As of now a total of eleven species (Hampson 1896;Kenrick 1907;Turner 1908;Caradja 1916;Meyrick 1937;Caradja & Meyrick 1937;Kitching et al. 2020) are considered extant in the genus of which, five species are reported from India (Kitching et al. 2020). Hampson (1896) mentioned the distribution of P. tyres as throughout India (having type locality in the Coromandel region of southern India).
For easy identification of the morphological cryptic species, mt DNA barcoding are being used as an alternative tool for insect species identification and documentation of new species (Hebert et al. 2003). Although DNA barcode-based species identification works are in infancy in the developing countries, the technique provides robust and rapid approach for biodiversity analysis (Ashfaq et al. 2017), exploiting low conspecific and high interspecific genetic variation principle (Hebert et al. 2003). DNA barcodes have been constructively utilized for diverse aims in addition to serving as an aid to conventional slow-paced taxonomic delimitation approaches (Ashfaq et al. 2017). DNA barcodes having effectively applied to unpin species identity for numerous animal taxa, the order Lepidoptera has seen particularly intensive barcode analysis (Ashfaq et al. 2017). The identification using DNA barcoding approach exclusively depends on the quality of reference library, which is strengthened if the barcodes are linked to registered voucher specimens. Identification of moths using mt DNA barcode has been introduced in the moth groups of Olepa (Kalawate et al. 2020a,b). Despite its widespread distribution there are no genetic data available for the species from India. Hence, during one of our exercise of generation of mt DNA barcodes for the moth species, here we report the first mt DNA barcode for the species P. tyres from India, having a wide range of distribution.

Materials and Methods
The specimens were collected by installing light trap during night, and were euthanized by ethyl acetate vapours. The specimens were transferred to the laboratory in insect packets under dry conditions. They were stretched, pinned, labelled, and dry-preserved in fumigated entomological boxes for further study. For morphological studies the specimens were studied under Leica EZ4E stereomicroscope. The map of the collection locality was prepared using open free QGIS software. The details of collection locality are given under material examined and also shown in Figure 1. The identification was done with the help of Hampson (1896). The genitalia of male and female were studied following Robinson (1976). The identified materials are deposited at the National Zoological Collections of the Zoological Survey of India, Western Regional Centre, Pune, Maharashtra, India (ZSI, WRC). DNA extraction and purification were performed using leg and thoracic muscle from dried specimen, followed by quantitation utilizing HS dsDNA assay kit on Qubit 2.0 fluorometer. Amplification of mt COI gene was attempted using universal primer (Folmer et al. 1994), LCO1490 and HCO2198 in 25µL reaction volume constituted by 12.5 µL of Master Mix (Promega), 10 pmol of each forward and reverse primer along with Nuclease free water up to Q.S. thermal cycling profile as per Kalawate et al. (2020a). Amplified PCR product was confirmed by gel electrophoresis stained by SYBR safe DNA gel stain (Invitrogen), visualized under UV by gel documentation system, followed by purification of amplified product by Invitrogen's Pure Link PCR Purification Kit. Purified PCR product was sequenced bidirectionally by Sanger's method on ABI 377 (Applied Biosciences) sequencer.
Both the forward and reverse sequences generated in the current study were verified manually for corrections. From the GenBank 21 mt COI gene sequences available for the Pygospila were downloaded (Table 1)

Result and Discussions
Morphologically the collected samples were identified as Pygospila tyres (Cramer, 1780) (Image 1).

Taxonomic account
Superfamily Hindwing with nacreous streaks in and below the cell. A pair of spots present between origin of vein 3 and 5, three submarginal spots and a spot present below vein 2; cilia brown and white towards anal angle. Underside exactly same pattern on both fore and hindwings. Hind wing of male with vein 8 widely separated from 7, 6 bent downward, the veins beyond the cell roughly scaled.

DNA Barcode diagnosis
The genetic sequence of sample of P. tyres from Pune, Maharashtra matches completely with the P. tyres sequences from Pakistan, Korea, and Australia. The clade composing the P. tyres is homologous without any genetic distance variation. One of the sequences (JX017862.1) from Australia is labelled as P. tyres, where the identity should be rechecked with the voucher

Image 1. Pygospila tyres: A-Adult | B-Male genitalia | C-Aedeagus | D-Female genitalia.
specimens as the sequence is forming monophyletic clade with the members of P. bivittalis from Australia. Although there are limitations with the phylogenetic inferences of mt COI DNA barcode trees, our studies could discern three clear clades for the species P. tyres, P. bivittalis and P. hyalotypa. Of the extant eleven species of Pygospila, we could include data of three species in the phylogenetic studies including our sequences from India for P. tyres.
Since the species P. tyres is of economic importance, the present mt DNA Barcode data generated is expected to be helpful in building a reliable DNA barcode library for the country intimated with a voucher specimen and helpful in addressing the taxonomic problems as the morphological characters are cryptic. Interestingly P. tyres was described almost 240 years ago from India J TT and now the species is known to have a wide range of distribution in Asia and Australia. Original description of the species P. tyres from Coromandel region and our multiple collections from the parts of Deccan plateau and the northern Western Ghats are similar in morphological characters. Genetic homogeneity with mt COI DNA gene studies across the two continents (Asia and Australia) reestablishes the wide distribution across these landscapes. The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.