First record and DNA barcode of a scarab beetle, Adoretus kanarensis Arrow, 1917 (Coleoptera: Scarabaeidae: Rutelinae), from Maharashtra, India

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ISSN 0974-7907 (Online); ISSN 0974-7893 (Print)  2011). There are about 460 species (Krajcik 2007) reported in the world, of which 47 are reported in India (Arrow 1917), few literature is available on this genus from India (Chandra 2009;Chandra et al. 2012;Ghosh et al. 2020). Some of the species of this genus are of biosecurity concerns (McQuate & Jameson 2011). The accurate identification based on the morphological characters are important for undertaking proper control measures. Beetles of the subfamily Rutelinae are not dung beetles in the true sense. They are phytophagous and commonly known as May or June beetles or shining leaf chafers (Sreedevi et al. 2017). The pioneering work on this group in India was undertaken by Arrow (1917) and Balthasar (1963Balthasar ( , 1974 Though Adoretus species are widely distributed, only 12-13 mitochondrial cytochrome oxidase subunit (COI) sequences are currently available in global database. Hence, during one of our experiments to generate mt DNA barcodes for the coleopteran species, we report the first mt DNA barcode of A. kanarensis from Maharashtra, India.

Sampling of dung beetles
Specimen for the present study was collected at night using light trap. 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.

Preservation and Identification
The collected specimen was euthanized in the vapours of ethyl acetate and brought to the laboratory for further studies. For morphological identification, the specimen was studied under Leica EZ4E stereomicroscope. The identification was done following the keys of Arrow (1917). Further, the voucher specimen was deposited in the National Repository of Zoological Survey of India, Western Regional Centre, Pune, Maharashtra (India).

DNA isolation, PCR and Sequencing
The ethanol preserved tissue was used for DNA isolation. DNA from the tissues of the beetle was extracted from metathoracic leg using DNeasy kit (Qiagen), according to the manufacturer's protocol. The obtained DNA was amplified using polymerase chain reaction (PCR) using ABI thermocycler. Following primers (Meyer et al. 2005) were used for amplification of COI gene: dgLCO F1 5'GGTCAACAAATCATAAAGAYATYGG 3' and dgHCO R1 5'TAAACTTCAGGGTGACCAAARAAYCA 3'. PCR reaction was carried out in total volume of 25 µl containing 2 µl DNA template, 10 pmol of each primer and 2 µl of dNTP and 0.2 µl of Taq polymerase (Bangalore GeNei). Thermo-cycling conditions were as follows: One initial cycle of 1 min at 95˚C followed by five cycles of 95˚C for 1 min, then denaturation 35 cycles of 95˚C for 1 min, annealing at 52°C for 40 s, extension at 72˚C for 1 min 15 s, with final extension of 72˚C for 5 min.
From each PCR reaction, 2 μL of the PCR product was visualized on a 2% agarose gel stained with ethidium bromide, together with a GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific). The obtained PCR products were sequenced with both, the forward and reverse, primers using an automated sequencer (3730 DNA analyzer, ABI, Hitachi).

Data analysis
Sequence was edited to remove ambiguous base calls and the forward and the reverse sequences were assembled using Chromas Pro version 1.34 (Technelysium Pty Ltd., Tewantin, Queensland, Australia). FASTA format of Adoretus kanarensis sequences was used for performed BLAST search at NCBI and species identification tool at Barcode of Life Data System (BOLD). All the obtained sequences were aligned and manually edited using BioEdit version 7.2.6. The Maximum Likelihood method and General Time Reversible model (GTR) model of base substitution was used to calculate pairwise genetic distance in MEGA X version 10.0.5. Additionally, to check the performance of DNA barcoding, sequences were downloaded from NCBI and BOLD (Table 2, Supplementary data) for some species of same genus submitted from different geographical areas. Only sequences which formed monophyletic clades with the sequences studied here were selected, to avoid use of sequences from wrongly identified species. These sequences along with our data were used to generate trees using MEGA X (Nei & Kumar 2000;Kumar et al. 2018).

Results and Discussions
Morphologically, the collected sample was identified as Adoretus kanarensis Arrow, 1917 (Figure 3).

Diagnosis
Female (Image 1): Length, 10 mm; width, 5 mm. Bright brownish-yellow, moderately shining. The lateral margins of head, pronotum, broad sutural line reaching anteriorly till the humeral callus and posteriorly not reaching the margins and extremities of tibia and complete tarsus are dark reddish-brown. Head transversely rugose, small, with the clypeus broadly rounded. The pygidium has a bare apical area. Legs are slender, the front tibia is armed with three strong teeth, the larger claw of the front and middle feet is cleft, and the shorter hind claw is more than half the length of the longer.

DNA Barcode diagnosis
In this research study, A. kanarensis was identified using available literature and sequence of COI gene isolated from an adult female. No matches were found among the already-existing entries in the BOLD database after analysis with the BOLD Identification tool. The genetic difference between the two samples was over 10%, indicating that the examined species has not yet been recorded in BOLD. GenBank's BLAST analysis yielded the same outcome.
The preliminary molecular analysis was carried out using available material from NCBI GenBank ( Since the species A. kanarensis is of economic significance, 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.

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