First report of the beetle Henosepilachna nana (Kapur, 1950) (Coleoptera: Coccinellidae) from Maharashtra with special reference to molecular phylogeny and host plants

: A ladybird beetle, collected from different localities of Kolhapur and Satara districts (Maharashtra) was identified as Henosepilachna nana (Kapur, 1950). The presence of this species in Maharashtra considerably extends its range to the north-west by about 700 km. Since this species is found on vegetables, Pumpkin ( Cucurbita spp.), Cucumber ( Cucumis spp.), and Karit fruit plant ( Cucumis spp.). It is being recorded for the first time that this species is a pest of these vegetables. Also, molecular phylogeny has been studied for the first time in this species in which, this species is the sister taxon of Henosepilachna boisduvali . This species has been described briefly with colour photographs of male genitalia, female coxites and the 6 th sternal plate of female, tarsi, & pronotum. Henosepilachna nana is being reported for the first time from Maharashtra and now the molecular data of this species is available.

The genus Epilachna is one of the genera of the subfamily Epilachninae in which hundreds of species from all over the world have been described and later many species were subsequently removed to other genera (Jadwiszczak & Węgrzynowicz 2003). Based on basal toothed tarsal claw and longitudinally divided sixth female abdominal sternite, Li & Cook (1961) raised a separate genus Henosepilachna from Epilachna. To date, there are about 110 described species of the genus Henosepilachna from Asia and Australia (Szawaryn 2011). There are 33 species of Henosepilachna in India (Poorani 2012;Poorani et al. 2021).
The male specimen collected from Nilgiri Hills, India has been described by Kapur (1950) as E. nana and its holotype is in the Zoological Survey of India (No. 3426/13). In the world catalogue of Coccinellidae, Jadwiszczak & Wegrzynowicz (2003) placed E. nana in the genus Henosepilachna, which is followed by Poorani (2012) in the updated checklist of Coccinellidae from Indian subregion and Borowski (2020) in the inventory of world Epilachninae. In the present study, we describe the detailed morphology of the male genitalia, 6 th sternal plate, female genitalia and tarsi of H. nana along with coloured photographs. In addition, this species is being reported for the first time in Maharashtra through this communication and its molecular phylogeny has been given.

MATERIAL AND METHODS
Specimens of H. nana were collected on different vegetable crops by handpicking method from Halondi (Hatkanangale), Kanthewadi (Radhanagari) and Parite (Karveer) of Kolhapur district and Sajjangad and Yerad (Patan) of Satara district of Maharashtra, India (Image 1). Specimens were photographed on stereo zoom microscope LYNX LM-52-3621 using TCapture software and preserved dry and wet and deposited at the Department of Zoology, Shivaji University, Kolhapur. Captured images stacked in Helicon Focus 7 software. Measurements were taken in ImageJ software. The genitalia was dissected and photographed using the same microscope and software. Species was identified and confirmed based on the detailed taxonomic description provided by Kapur (1950) along with genitalia description.

Molecular analysis
Genomic DNA extraction was done by the manual CTAB method (Boopathi et al. 2020). PCR of extracted DNA was carried out using the primers LepF1-5'-ATTCAACCAATCATAAAGATATTGG-3' and LepR1-5'-TAAACTTCTGGATGTCCAAAAAATCA-3' (Hebert et al. 2004;Wilson 2012). PCR amplified mitochondrial COI gene was sequenced by the Sanger sequencing method. The obtained sequence were submitted to GenBank. To construct the phylogenetic tree, sequences for the COI gene of Henosepilachna were downloaded from NCBI. A total of 54 sequences for eleven species of Henosepilachna and one for Epilachna were downloaded from NCBI GenBank (Table 1) of which Epilachna sp. is an outgroup.
Generated COI gene sequences were edited in BioEdit Sequence Alignment Editor Software. Multiple sequence alignment was carried out using MUSCLE (Edgar 2004) in MEGA 7 (Kumar et al. 2016). Phylogenetic analysis was done for COI sequences for 13 taxa.
Phylogenetic analysis was carried out using Maximum Likelihood (ML) method (Felsenstein 1981). MEGA 7 was used to find out the best-fit nucleotide model for the dataset under the corrected Akaike Information Criterion (AICc). The best nucleotide substitution found was T92+G, which is not implemented in the RAxML program, so we used the default model which was GTR + G (Lanave et al. 1984;Gu et al. 1995). ML analysis was performed on RAxML HPC2 (Stamatakis 2014) at the CIPRES portal (Miller et al. 2010). Bootstrap support values were obtained with the rapid bootstrap algorithm with 1000 bootstrap replicates.

Henosepilachna nana (Kapur, 1950)
Diagnostics: Length 6.0-6.6 mm, width 4.6-5.2 mm. The body is oval with 6 black elytral spots over grey background (Image 2a). The black elytral spots are surrounded by a brown ring. The dorsal body shows grey pubescence except for black spots. Brick red coloured pronotum with four black spots (Image 2m). In many examples, pronotal spots on the same side join to form a quadrate spot, which is nearer to the base than the apex and does not touch to anterolateral margins and pronotal median line (Image 2l). Scutellum brown, triangular, pointed at the elytral side (Image 2i). Ventrally brown except dark patches on metasternum and abdominal sternites. Tarsi are pseudotrimerous having bifid tarsal claws (Image 2o). The tarsal claw is with a basal tooth (Image 2p). Sixth abdominal sternite is divided in females (Image 2h ) and emarginated in males (Image 2j).
Male genitalia: Penis is thin, with a slightly broad penis capsule (Image 2f). Penis apex is with V-shaped notch and a small projection at the notch base (Image 2d,e). Tegmen with paramere tip rounded having many setae at the apex and fewer setae on the distal half. Penis guide is uniformly curved at the distal half, curved and pointed at the apex (Image 2b,c).
Female genitalia: Female coxites quadrate with blunt corners, inner margin is straight except for a shallow notch at the base and with a pear-shaped small stylus with numerous setae (Image 2g).
Host plants: The specimens of Henosepilachna nana were found to be associated with Cucumber (Cucumis spp.), Pumpkin (Cucurbita spp.), and Karit fruit plant (Cucumis spp.). On the underside of leaves, in a bunch, approximately 20 elliptical, yellow, sparsely laid eggs were observed. Both the larvae and adults of H. nana skeletonize the leaves of the above vegetables by eating the chlorophyll and can act as a serious pest Phylogeny (Figure 1) Gordon (1975) revised Epilachninae of the Western hemisphere in which he described one new species named E. nana. However, the species which was described by Gordon (1975) is different from E. nana having different characteristics than E. nana which was described by Kapur (1950). Later, Kapur informed Gordon that the nomenclature E. nana is already given to one ladybird species and then Gordon (1985) replaced the E. nana name with E. minuta. The Holotype of E. nana was collected in 1892 and paratypes were collected in 1914 from Nilgiri Hills and Parambikulam (Kerala), respectively (Kapur 1950). The species is distributed over the Nilgiri Hills of southern India (Kapur 1950;Poorani 2012;Borowski 2020). The localities in Maharashtra, which are being reported in the present article, will add H. nana to the Fauna of Maharashtra, proving that the range extension of this species towards the north-west by about 700 km.
According to the illustrations and description given by Kapur (1950), the specimens recorded during the study are treated here as H. nana. Every specimen of H. nana collected during the study show 12 elytral spots without any variation in the count. Based on the number of elytral spots, H. nana differs from the other Henoesepilachna species. The species H. boisduvali is yellowish-red having a median black pronotal spot and 12 elytral spots (Li & Cook 1961;Li 1993). The species H. nana is varying from H. boisduvali in having two spots present on each side of the median line, there is no median pronotal spot and the median area is always spotless. In the male genitalia, the penis tip of nana. Therefore, the COI gene sequence of H. nana is becoming available in the NCBI database for the first time. The present consensus tree arrangement shows the co-evolution of species from common ancestors. When the phylogenetic tree of H. nana was constructed using COI gene sequences of eleven species of the Image 2. a-Henosepilachna nana habitus | b-male genitalia: tegmen lateral view | c-tegmen ventral view | d-penis tip-lateral view | e-penis tip-ventral view | f-penis | g-female coxites | h-6th abdominal sternite of female | i-scutellum | j-6th abdominal sternite of male | k-mandible | l,m-pronotum | n-feeding adults | o-tarsi | p-tarsal claw. © Priyanka Patil.

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