Molecular Phylogeny of Scymnus Latifolius, a Predator Species of Mealy Bug Shows Divergent Evolution Among Scymnus Species

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13. Ghose, N. C., In Recent Researches in 22. Saha, A., Ganguly, S., Ray, J. S. and ACKNOWLEDGEMENTS. We thank the Geology (ed. Sinha-Roy, S.), Hindustan Dhang, A., J. Geol. Soc. India, 2010, 76, Head, Department of Geology, BHU, Varanasi Publishing Corporation, Delhi, 1983, vol. 26–32. for the departmental support. N.V.C.R. thanks 10, pp. 211–247. 23. Banerjee, P. K., Chem. Geol., 1984, 43, DST-SERB for granting research project 14. Mukhopadhyay, D., Precambrian of the 257–269. (IR/S4/ESF-18/2011 dated 12.11.2013). D.K. Eastern Indian Shield, Geological Society 24. Spencer, K. J. and Lindsley, D. H., thanks CSIR, New Delhi for the award of JRF of India Memoir, 1988, 8, 237. Am. Mineral., 1981, 66(11–12), 1189– (NET) and SRF. We also thank Dr Ajit K. 15. Naqvi, S. M. and Rogers, J. J. W., Pre- 1201. Sahoo (BHU) for useful discussions. Con- cambrian Geology of India, Oxford 25. Carmichael, I. S. E., Contrib. Mineral. structive review by an anonymous journal Univ. Press, 1987, p. 223. Petrol., 1967, 14(1), 36–64. reviewer is thankfully acknowledged. 16. Kumar, A. and Ahmad, T., Geochem. J., 26. Mohanty, J. K., Khaoash, S., Singh, S. 2007, 41, 173–186. K., Sahoo, P. K. and Paul, A. K., India Received 1 December 2020; revised accepted 17. Sharma, R. S., Cratons and Fold Belts of Scan. J. Metal., 1999, 28(6), 254–259. 12 January 2021 India, Springer-Verlag, 2009, p. 304. 27. Devaraju, T. C., Viljoen, R. P., Sawkar, 18. Srivastava, R. K., Kumar, S. and Sinha, R. H. and Sudhakara, T. L., J. Geol. Soc. A. K., J. Earth Syst. Sci., 2012, 121, India, 2009, 73, 73–100. DEEPAK KUMAR 509–523. 28. Acharyya, S. K., Gondwana Res., 2003, DINESH PANDIT 19. Srivastava, R. K., Kumar, S., Sinha, A. 6(2), 197–214. N. V. CHALAPATHI RAO* K. and Chalapathi Rao, N. V., J. Asian 29. Ray, J. N., Geological Survey of India Earth Sci., 2013, 84, 34–50. map of Raurkela Quadrangle – Bihar, Department of Geology, 20. Chalapathi Rao, N. V., Srivastava, R. K., Madhya Pradesh and Orissa. Toposheet Institute of Science, Sinha, A. K. and Ravikant, V., Earth Sci. number 73B, 1983. Rev., 2014, 136, 96–120. 30. Ondrejka, M., Broska, I. and Uher, P., Banaras Hindu University, 21. Kumar, D., Pandit, D., Sharma, A. and Acta Geol. Slovaca, 2015, 7(1), 51–61. Varanasi 221 005, India Chalapathi Rao, N. V., Curr. Sci., 2019, 31. Schuiling, R. D. and Feenstra, A., Chem. *For correspondence. 117(5), 858–865. Geol., 1980, 30, 143–150. e-mail: [email protected]

Molecular phylogeny of Scymnus latifolius, a predator species of mealy bug shows divergent evolution among Scymnus species

Ladybird beetles belong to the family present study was conducted to identify National Center for Biotechnology Coccinellidae, super family Cucujoidea, the phylogenetic relationship of Scymnus Information (NCBI) and can be accessed suborder Polyphaga of order Coleoptera1, species with other Scymnini and cocci- at GenBank (accession number KU512906). and consist of more than 360 genera and nellids through comparative analysis of Sequence diversity of specific 595 bp 6000 species2. Many of the coccinellid partial sequences of mitochondrial COI fragment of the mitochondrial COI gene insects are widely used as predators in gene. (the COI 5′ region) amplified from S. la- the biological control of major agricul- Insect collections were obtained from tifolius was compared with COI gene se- tural pests. Previously, we have descri- mulberry gardens located in Murshida- quences of 44 different Coccinellids with bed a novel predator, Scymnus (Pullus) bad, Malda, Birbhum and Nadia districts, one out-group from another subfamily latifolius Poorani belonging to Scymnini West Bengal, India using standard tech- (Serangium spp.) available in the NCBI tribe of the family Coccinellidae3. The S. niques. Adult specimens of S. latifolius database. The initial multiple alignment latifolius beetle is found to predate upon were positively identified using morpho- and sequence editing were carried out all developmental stages of several mea- logical descriptions2 and preserved in using Molecular Evolutionary Genetics lybug species, a major pest with a wide 85% ethanol in the dark at 4°C until fur- Analysis software (MEGAX)12. Later, host range, and hence could play a key ther analysis. DNA was isolated from the phylogenetic analysis was carried out role in biological control. hind legs of individual beetles using a employing Bayesian approaches13 using Several phylogenetic subdivisions for DNA isolation kit (Qiagen, Germany) Mr Bayes programme (Mr Bayes 3.2.7v the family Coccinellidae have been pro- following the manufacturer’s protocol WIN64) available at https://nbisweden. posed based on conventional morpho- and stored at –20°C until use. Polyme- github.io/MrBayes/download.html. Before logical observations2,3 and molecular rase chain reaction (PCR) amplification constructing the phylogenetic tree, methods4,5. Mitochondrial gene sequen- of partial gene sequences of mitochon- the substitution model was analysed ces have been widely used to understand drial COI gene was conducted using the using MEGAX software. The model the evolutionary history of coleopte- universal COI primers10 following a GTR + I + G was found to fit well with rans6,7 and coccinellids in particular4,5,8,9. method described previously11. The PCR lowest Bayesian inference (BI) index. BI The mitochondrial cytochrome C oxidase products were purified using a kit (Qia- analyses were done using two runs simul- subunit I (COI) gene nucleotide sequen- gen, Germany) and sequenced by San- taneously, with maximum likelihood ces have been extensively used for phy- ger’s method at a commercial facility. starting tree, and four chains were used logenetic analysis and species-level The nucleotide sequences of COI gene for the analysis (one cold and three hot) identification of Coccinellidae9. The generated have been submitted to the and the temperature set at 0.1. A run was

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Figure 1. Bayesian inference tree with 50% majority-rule consensus. The posterior probability is given at each node of various Scymnus species. Serangium spp. is used as outgroup. set for 10 million generations with sam- results of the present study are in agree- agreement with earlier observations that ple frequency of 1000. The initial 25% ment with previously described molecu- the subfamily Scymninae, the genera trees were discarded using Burn-in 25%, lar phylogeny of coccinellids4,5,14. Based Nephus, Scymnus and Diomus are con- and a 50% majority rule consensus tree on branch length, the species Micrapsis sidered monophyletic with different was constructed from the remaining trees discolor can be considered to be more lineages within the subfamily, with Ne- and posterior probabilities were esti- diverged from the rest of the species and phus and Scymnus spp. as sister taxa5. mated for each nodes. The final consen- has close association with Cryptoleamus Even though the evolution of different sus tree was built using FigTree1.4v montrouzieri. However, a detailed study species of Scymninae, Coccinellinae, available online (http://tree.bio.ed.ac.uk/ is necessary to explain their evolutionary Chilocorinae and Sticholotidinae could software/figtree/) by 50% majority-rule relationship. not be specifically described, a clear consensus. Figure 1 shows the branch The consensus phylogenetic tree is trend of mixed lineages could be seen length and posterior probability for each suggestive of S. latifolius as a different among these subfamilies, suggesting a node calculated and the consensus tree species, supporting the conventional polyphyletic origin. However, the generated. morphological study3. As can be obser- present study does not find supporting The analysis suggests a close phylo- ved from the Bayesian inference phylo- results against the monophyletic origin genetic association of S. latifolius with genetic tree, the genus Scymnus evolved of Scymninae, which needs further exa- other Scymnus spp. and coccinellids from as a single clade in the subfamily Scym- mination. the same subfamily included in the study. ninae, forming a single clade separated The morphological observations descri- In the phylogenetic tree, all the species from all other genera of the subfamily. bed earlier2 and molecular phylogenetic belonging to the genus Scymnus formed The genera Cryptoleamus and Micrapsis approaches were completely consistent the same clade with further few sub- form a single clade, which is paraphylet- with each other. The close evolutionary clades among themselves, whereas the ic to other genera such as Hyperapsis, relationship of S. latifolius with various other genera from the same subfamily Diomus and Nephus and Chilocorini Scymnini (Scymnus spp., Nephus spp. branched into major clades with a few (Chilocorus and Brumoides spp.). The and Cryptolaemus spp.), Diomini (Dio- subclades. The topology of the phyloge- consensus tree also suggest a co-lineage mus spp.) and Hyperapsidini (Hyperapsis netic tree was in line with the morpho- for Cryptolaemus montrouzieri along spp.) in Chilorinae, the tribe Chilocorini logical classification and broadly, the with S. latifolius. The present study is in (Chilocirus and Brumoides spp.) in

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Coccinellinae, the tribe Coccinellini Academic Publishers, Dordrecht, The ACKNOWLEDGEMENTS. We thank the (Coccinella, Micraspis, Harmonia, Chei- Netherlands, 1996. Central Silk Board for funds and laboratory lomenes, Anatis spp.) that was observed 3. Poorani, J. and Lalitha, N., Zootaxa, facilities. We also thank Dr Poorani Janaki- during morphological studies3 was fur- 2018, 4382, 93–120. raman (ICAR-NRC on Banana, Trichy) for 4. Magro, A., Lecompte, E., Magne, F., technical support in the identification of ther confirmed through sequence diversi- Hemptinne, J. L. and Crouau-Roy, B., Mol. insect specimens. This communication is part ty analysis of the COI gene. Phylogenet. Evol., 2010, 54, 833–848. of the Ph.D. thesis of first author submitted to In biological speciation, differences in 5. Seago, A. E., Giorgi, J. A., Li, J. and the Visva-Bharati University, Santiniketan. 4,15 shape of the genitalia is one of the Ślipiński, A., Mol. Phylogenet. Evol., most important natural mechanisms that 2011, 60, 137–151. results in lineage divergences and evolu- 6. Liu, H. and Beckenbach, A. T., Mol. Received 28 September 2020; revised tion in two or more descendant species16. Phylogenet. Evol., 1992, 1, 41–52. accepted 25 January 2021 Evolution of reproductive isolation with- 7. Song, H., Sheffield, N. C., Cameron, S. in an ancestral species causes genetic, L., Miller, K. B. and Whiting, M. F., 1,2,3, physiological and behavioural differenc- Syst. Entomol., 2010, 35, 429–448. N. LALITHA * 8. Escalona, H. E. et al., BMC Evol. Biol., 2 es that result in lineage divergences. The H. CHATTERJEE 2017, 17, 151. 4 presence of a unique male genitalia of R. GANDHI GRACY 9. Wang, Z. L., Wang, T. Z., Zhu, H. F., 1 Scymnus latifolius that is different from Wang, Z. Y. and Yu, X. P., Mitochondri- M. V. SANTHAKUMAR other Scymnus species has been reported al DNA, Part A, 2019, 30, 1–8. earlier3. Thus, species divergence of S. 10. Hebert, P. D. N., Penton, E. H., Burns, J. 1Central Sericultural Research and latifolius observed in the molecular M., Janzen, D. H. and Hallwachs, W., Training Institute, phylogenetic analysis is supported by Proc. Natl. Acad. Sci. USA, 2004, 101, Central Silk Board, morphological studies3. To conclude, a 14812–14817. Berhampore 742 101, India lineage divergence of S. latifolius from 11. Ball, S. L. and Armstrong, K. F., J. 2Palli Siksha Bhavana, other Scymninae could be seen during Econ. Entomol., 2008, 101, 523–532. 12. Kumar, S., Stecher, G., Li, M., Knyaz, C. Visva-Bharati University, molecular phylogenetic analysis, con- and Tamura, K., Mol. Biol. Evol., 2018, Santiniketan 731 236, India firming its distinctiveness as a separate 35, 1547–1549. 3P2 Eri Silkworm Basic Seed Farm, species and its taxonomic position. Apart 13. Huelsenbeck, J. P., Larget, B. and Alfa- Central Silk Board, from the phylogenetic perspective, appli- ro, M. E., Mol. Biol. Evol., 2004, 21, Ministry of Textiles, cation of a suitable molecular marker 1123–1133. Topatoli, Guwahati, such as the COI gene can be useful to 14. Giorgi, J. A. et al., Biol. Control, 2009, Kamrup (M) 782 403, India identify insects during their morphologi- 51, 215–231. 4ICAR-National Bureau of Agricultural cally indistinct immature stages before 15. Lablokoff-Khnzorian, S. M., Les cocci- Insect Resources, emergence of the adults. nelles: Coléopterès-Coccinellidae: tribu Coccinellini des règions palèarctique et Hebbal, 1. Sasaji, H., Etizenia, 1968, 35, 1–37. orientale, Boubee, Paris, 1982. Bengaluru 560 068, India 2. Kovárˇ, I., In Ecology of Coccinellidae 16. Futuyma, D. J., Evolution, Sinauer, *For correspondence. (eds Hodek, I. and Honk, A.), Kluwer, Cambridge, MA, USA, 2009. e-mail: [email protected]

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