MALAYSIAN JOURNAL OF APPLIED SCIENCES 2019, VOL 4 (2): 10-18 E-ISSN:0127-9246 (ONLINE) https://journal.unisza.edu.my/myjas ORIGINAL ARTICLE Molecular Phylogeny of Predatory Ladybird Beetles (Coleoptera: Coccinellidae) Inferred from COI Sequences *Pisit Poolprasert 1, Sinlapachai Senarat 2, Sorasak Nak-eiam 3 and Natdanai Likhitrakarn 4 1Biology Program, Faculty of Science and Technology, Pibulsongkram Rajabhat University, Phitsanulok, 65000, Thailand 2Department of Marine Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 3Department of Biology, Faculty of Science and Technology, Rambhai Barni Rajabhat University, Chanthaburi, 22000, Thailand 4 Division of Plant Protection, Faculty of Agricultural Production, Maejo University, Chiang Mai, 50290, Thailand *Corresponding author: [email protected] Received: 21/05/2019, Accepted: 17/07/2019, Published: 31 December 2019 Abstract The Coccinellidae is a highly diversified family of the Coleoptera. Coccinellids are well recognized because of their use as biocontrol agents and they are the subject of many ecological studies. However, little is known concerning phylogenetic relationships of the Coccinellidae, and a precise evolutionary framework is still required for the family. In this study, therefore, we endeavoured to use molecular taxonomic identification on the Cocinellidae. We also provided the first phylogenetic reconstruction of the relationships within the Coccinellidae based on analysis of a 658-bp fragment of mitochondrial DNA from the 5’ region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Afterwards, all obtained COI sequences were verified and compared to sequences previously deposited in Genbank using BLAST. It was discovered that they were similar to Coccinellidae with 99-100% identities. Eleven coccinellid species (10 genera) belonging to four subfamilies (Coccinellinae, Chilocorinae, Sticholotidinae and Scymninae) and relevant outgroup were reconstructed using the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates. The recovered phylogeny was revealed that most of subfamilies (except Sticholotidinae) of Coccinellidae were reciprocally monophyletic and the threshold level of nucleotide divergence was 0.155% ± 0.008, suggesting that this DNA barcoding, when combined with bioinformatics, provided a reliable method for the taxonomic classification of coccinellid beetles in this time. Keywords: Coccinellid beetles, COI gene, DNA barcoding, phylogenetic tree Introduction Nowadays, molecular technique based on DNA sequence can be applied to the taxonomic identification of identified and unidentified specimens (Hebert et al., 2003a, b, 2010). Even though several different DNA based techniques are potential available for quarantine and forensic MALAYSIAN JOURNAL OF APPLIED SCIENCES 2019, VOL 4 (2): 10-18 examinations (Yan et al., 2013). DNA barcoding, a modern method for the rapid identification of any species, has also been widely used as method to efficiently describe biodiversity (Wilson, 2012). Also, this technique use DNA fragments gained from specific genes, normally from mitochondrial DNA such as the cytochrome c oxidae I (COI) gene, a fragment of about 500-800 base pairs, has been extensively used in several animal species (Hebert et al., 2003a, b). Generally, it is presumed that mitochondrial DNA (mtDNA) evolves at a faster rate than nuclear DNA (nuDNA) in animals. This has contributed to the popularity of mtDNA as a molecular marker in evolutionary studies (Allio et al., 2017). In this regard, COI is more conserved and it is very suitable for species identification since its sequence has a low variability (less than 1-2%). Even for the closely-related species its value is less than 1%. In addition, COI gene is the most common gene used to analyze the relationship among closely-related species in several insect groups (butterflies, beetles and flies), as individual gene or its combination with other genes (Hebert et al., 2003a, b; 2010). Coccinellids, also known colloquially as ladybirds or ladybugs, belong to the insect family Coccinellidae and order Coleoptera and have received considerable study attention because of their potential as biological control agents. Indeed, they are one of the most vital groups of the natural predatory or enemy complex of several horticultural and agricultural crop pests i.e. scale insects, mealy bugs, aphids and other soft bodies arthropods (Atif et al., 2016; Escalona et al., 2017). The ladybird beetles are considered as having a great economic importance in agro- ecosystems through their successful biological control of numerous injurious insects (Seago et al., 2011). Coccinellidae are extremely diverse in their habitat and live in all types of terrestrial ecosystems from the plains to mountainous areas (Vandereycken et al., 2012, 2013; Verheggen et al., 2017). In addition, they are regarded as bioindicators and provide more general information about the ecosystem in which they exist (Halim et al., 2017). More than 6,000 distinct ladybird beetle species have been recorded globally. Meanwhile, over one hundred coccinellid beetles have been identified in Thailand (Chunram, 2002) The recently -discovered molecular phylogeny of the family coccinellidae (61 species and 37 genera based upon five genes e.g. the 18S and 28S rRNA nuclear genes and the mitochondrial 12S, 16S rRNA and COI genes) revealed that the Coccinellinae was monophyletic but the Coccidulinae, Epilachninae, Scymninae and Chilocorinae were paraphyletic (Magro et al., 2010). However, knowledge of phylogenetic relationships of Thai coccinellid beetles is rarely known. Therefore, the main purposes of this this study were to sequence mitochondrial cytochrome oxidase subunit I (COI) in and to evaluate former phylogenetic hypotheses regarding eleven ladybird beetle species belonging to four subfamilies i.e. Coccinellinae, Chilocorinae, Sticholotidinae and Scymninae under the family Coccinellidae exhibited from Thailand. Materials and Methods Coccinellid Sample Collection A total of eleven predatory ladybird beetle species were gathered from different vegetable crops i.e. cabbage, cauliflower, kale and lettuce in Phitsanulok and Sukhothai provinces, lower northern Thailand using yellow sticky traps together with hand picking and were subsequently preserved in 95% alcohol. All beetles were then identified morphologically to the genus and species levels using main published literatures of Chunram (2002), Giorgi and Vabdenberg (2012), Saeed et al. (2016) and Ali et al. (2018). DNA Extraction, Amplification and Sequencing Total genomic DNA was isolated from a single leg of coccinellids using DNeasy® Blood & Tissues kit (Qiagen, Germantown, MD, US, catalog #69504). The protein-coding mitochondrial COI gene for molecular analysis was used in this study. The primers used for the polymerase chain reaction (PCR) amplification were LCO1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and 11 MALAYSIAN JOURNAL OF APPLIED SCIENCES 2019, VOL 4 (2): 10-18 HCO2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) (Folmer et al., 1994). Each PCR reaction was performed using a final volume of 20 μl containing 4 μl of 5x PCR Enhancer, 2 μl of 10x HF Reaction Buffer, 0.4 μl 10 mM dNTP Mix, 0.3 of each primer (10 μmol/L), 0.3 μl of Long and High Fidelity DNA Polymerase (0.75 U) (biotechrabbit, Germany), 10.7 μl of nuclear free water and at least 2 ng of genomic DNA template. The cycling program included an initial activation step of 3 min at 94 ºC, followed by 35 cycles of 1 min at 94 ºC (denaturation), annealing temperature at 48 ºC for 1 min, extension at 72 ºC for 1 min and a final extension of 5 min at 72 ºC. The amplification products were visualized under UV light following electrophoresis on an ethidium bromide stained 1% agarose gel in 1x TAE buffer. A single band of PCR products was purified using the GenUP PCR/ Gel Cleanup Kit (biotechrabbit, Germany) as described by manufacture’s instruction and direct sequences by Macrogen, inc (http://www.macrogen.com). Alignment of sequences and Phylogenetic analysis Similarity search for each sequence was checked using BLAST (https://www.ncbi.nlm.nih.gov/). Partial COI sequences were initially aligned in MEGA6 v6.06 (Tamura et al., 2013) using ClustalW (1.6) with the default settings (Gap Opening Penalty = 15, Gap Extension Penalty = 6.66 in both pairwise and multiple alignments). All COI sequences were finally trimmed to 658 base pairs and deposited in GenBank under accession numbers MH187249-MH187260. Afterwards, phylogeny reconstructions were performed for Neighbor Joining NJ tree building method. Genetic distances were computed using Kimura’s 2-parameter (K2P) test. The statistical confidence of a particular clade in the tree building method was evaluated by using bootstrap test with 1,000 replications. Additionally, overall AT bias and nucleotide diversity (π ± SD.) were calculated using DNAsp v5.10.01 program (Librado and Rozas, 2009). Results and discussion The DNA sequence of the mitochondrial 5'COI gene region was isolated to infer phylogenetic correlation of the lady beetles (family Coccinellidae). DNA fragments containing 658 base pairs of COI gene were successfully obtained from 11 coccinellid species including Coccinella transversalis, Coleophora inaequalis, Harmonia octomaculata, Menochilus sexmaculatas; Micraspis discolor, Brumoides sp., Chilocorus politus, Chilocorus nigritus, Pharoscymnus sp., Nephus sp. and Scymnus sp. belonging to four subfamilies (Coccinellinae, Chilocorinae,