Coleoptera: Coccinellidae) Inferred from COI Sequences

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 4Division 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

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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, Sticholotidinae and Scymninae) (see Figure 1). Consequently, the transition/transversion rate ratios are k1 = 1.097 (purines) and k2 = 2.478 (pyrimidines). The overall transition/transversion bias was R = 0.796, where R = [A*G*k1 + T*C*k2]/[(A+G)*(T+C)]. The average nucleotide composition proportions for all coccinellid sequences were detected to be A = 31.60% (30.70- 32.8%), T = 38.50% (36.50-39.70%), G = 14.70% (13.70-15.80%) and C = 15.20% (13.20- 16.70%). However, the overall frequency distributions of nucleotides at the 1st, 2nd and 3rd codon positions were as follows: A = 48.20%, 32.20% and 14.30%; C = 6.60%, 14.60% and 24.30%; G = 2.70%, 24.90% and 16.60% and T = 42.0%, 28.0% and 45.0%, respectively. These findings conformed to the results of the COI gene region of other insect groups exhibiting a bias towards adenine and thymine which was consistent with the base composition of the corresponding COI region. Generally, the base composition of the COI fragment varied among the species but it was commonly demonstrated with an overall AT bias of 66.70% and 33.3% (Jing and Yingchun, 2006). Correspondingly, the AT and GC contents for these results were 70.10% and 29.90%, respectively. However, they slightly differed from using the sequences of the 16S rDNA gene to infer phylogenetic relationships within the Coccinellidae. The average nucleotide composition proportions for the Coccinellidae sequences were: G, 14.5; A, 37.6; T, 40.6; and C, 7.3, implying that AT content (78.20%) was at a higher level (Aruggoda et al., 2010). To estimate interspecific

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divergence, genetic distances among 11 coccinellids were calculated using the net between the group average using MEGA6 v6.06. The interspecific variation in the base composition in COI displayed very low for the total nucleotides (0.15490% ± 0.00831). Besides, the genetic divergence for the coccinellid species ranged from 0.11 (11%) (Brumoides sp. and Pharoscymnus sp.) to 0.26 (26%) (Scymnus sp. and Cheilomenes sexmaculata) as demonstrated in Table 1.

Figure 1: Morphological diversity of Coccinellidae, habitus view of adults. (A) Coccinella transversalis; (B) Coleophora inaequalis; (C) Harmonia octomaculata; (D) Menochilus sexmaculatas; (E) Micraspis discolor; (F) Brumoides sp.; (G) Chilocorus politus; (H) Ch. nigritus; (I) Pharoscymnus sp.; (J) Nephus sp. and (K) Scymnus sp., respectively (Scale bar = 1 mm)

Table 1: Genetic distance (K2P) (lower diagonal) and standard deviation values (upper diagonal) obtained from COI gene sequence analysis

Ladybeetle Species (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) Nephus sp. (1) 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 Chilocorus nigritus (2) 0.14 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 Chilocorus politus (3) 0.17 0.14 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 Brumoides sp. (4) 0.14 0.14 0.15 0.02 0.02 0.02 0.03 0.02 0.02 0.02 Pharoscymnus sp. (5) 0.18 0.14 0.20 0.11 0.02 0.02 0.03 0.03 0.02 0.02 Harmonia octomaculata (6) 0.19 0.19 0.20 0.16 0.17 0.02 0.02 0.02 0.02 0.02 Coccinella transversalis (7) 0.23 0.23 0.21 0.20 0.22 0.18 0.02 0.02 0.02 0.02 Micraspis discolor (8) 0.24 0.21 0.23 0.23 0.29 0.23 0.17 0.02 0.03 0.03 Cheilomenes sexmaculata (9) 0.25 0.20 0.24 0.21 0.23 0.22 0.22 0.23 0.03 0.03 Coelophora inaequalis (10) 0.21 0.20 0.22 0.16 0.20 0.19 0.17 0.24 0.22 0.02 Scymnus sp. (11) 0.17 0.16 0.19 0.16 0.19 0.19 0.19 0.24 0.26 0.19

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The phylogenetic relationship of the eleven coccinellid species (10 genera) belonging to four subfamilies (Coccinellinae, Chilocorinae, Sticholotidinae and Scymninae) under the family Coccinellidae and relevant outgroup (Family Chysomelidae) was reconstructed using the Neighbor joining (NJ) method with 1,000 bootstrap replicates. With regards to phylogenetic analysis, it demonstrated as a single mtCOI clade for NJ. The recovered phylogeny revealed that most lineages subfamilies (except Sticholotidinae) of Coccinellidae were reciprocally monophyletic (Figure 2). This result was closely agreed with Seago et al. (2011) who proposed an evolutionary classification of the ladybird beetles (family Coccinellidae). In the same manner, Aruggoda et al. (2010) addressed the phylogeny of Coccinellidae inferred from 16S rDNA fragments. It indicated that 16S rDNA phylogenetic analyses were supported well on below tribe level relationships of the subfamilies. Moreover, based on COI, COII, 28S, it was found that the subfamily Coccinellinae was monophyletic. The Chilocorinae, Scymninae and Coccidulinae formed a second closely related cluster (Nedved and Kovar, 2012). COI discriminated well at the intergeneric, intraspecific and interspecific levels (Palenko et al., 2004). Worldwide, seven subfamilies of the Cocinellidae; Chilocorinae, Coccidulinae, Coccinellidae, Epilachninae Ortaliinae Scymninae and Sticholotidinae have been documented (Roy and Migeon, 2010; Halim et al., 2017). Meanwhile, Thai ladybeetle species are represented by only six subfamilies including Chilocorinae, Coccidulinae, Coccinellidae, Epilachninae, Scymninae and Sticholotidinae (Chunram, 2002). In this study, representatives from only four subfamilies of predatory coccinellids were employed for phylogenetic test due to no samples of Coccidulinae were collected and most specimens in the Epilachninae were phytophagous. Previous analyses supported the polyphyly of Scymninae, Coccidulinae and Chilocorinae and provided for the first time evidence of the polyphyly of Epilachninae (Magro et al., 2010). Nevertheless, the subfamilies Sticholotidinae, Chilocorinae, Scymninae, and Coccidulinae could be illustrated as paraphyletic in some case. (Girogi et al., 2009). Thus, both subfamilies as mentioned above remain to be sufficient phylogenetic constructed. Besides, in the case of Sticholotidinae in this study, its monophyly was not rejected, but inadequate sample to support the phylogenetic construction. Therefore, more samples of the Sticholotidinae are required for further phylogenetic status. From this current research, it established that DNA barcoding was an effective yardstick for rapid, reliable genus and species-level identification of coccinellid beetles. Other researches, focusing, for example on Lepidoptera (butterflies and moths), Hymenoptera (bees and wasps), Neuropterida (lacewings and antlions), Heteroptera (true bugs), Myriapoda (millipedes, centipedes, pauropods, and symphylans) and Orthoptera (grasshoppers and crickets) have obtained similar results, strongly suggesting the efficacy of DNA barcoding for all arthropods (Hebert et al., 2003a, b, 2010; Wilson, 2012; Karthika et al., 2016). Nonetheless, to obtain distinctly data in terms of phylogenetic status of Thai coccinellidae and to be useful for further agricultural managements, analysis of tree topology based on both mitochondrial and nuclear genes should be initially elucidated.

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Figure 2: Phylogenetic tree of the COI gene sequences inferred based on analysis of COI with NJ showing the position of the representatives of different subfamilies of Coccinellidae and two relevant species i.e. Cassida rubiginosa and Phaedon brassicae (F. Chrysomelidae) served as outgruop. Species names are followed by collection locality (AT: Austria, CA: Canada, CN: China, DE: Germany, FR: France, IN: India, MYS: Malaysia, PK: Pakistan, TH: Thailand and US: United States of America).

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Conclusion

In brief, this current analysis represented the first molecular phylogeny of the predatory coccinellids based on Thai specimens. Regarding the DNA sequencing, it was found that most coccinellid samples could be well classified into species level, suggesting that COI mtDNA sequencing is a suitable method for rapid species identification and could be applied for other insect groups. The eleven coccinellid species belonging to family Coccinelidae was recovered as monophyletic in this analysis. Nonetheless, the tests of monophyly for the subfamilies Coccinellinae, Chilocorinae, Sticholotidinae and Scymninae were relatively weak due to the small and unrepresentative taxon samplings that were available. The lack strong support for interfamilial relationships demonstrated that these data alone were inadequate to clearly resolve relationships among coccinellid subfamilies. However, future research which builds on this study should provide additional insight regarding the relationships among these enigmatic lineages and establish a foundation for a more natural and stable classification for this insect group.

Acknowledgments

This research was financially supported by the Thailand (NRCT) and the Higher Education Research Promotion (HERP). The authors would like to thank Professor Dr. David Haymer, University of Hawaii for his valuable comments on this work. We also thank the Faculty of Science and Technology, Pibulsongkram Rajabhat University for providing facilities and laboratory supports. Special thanks to Mr. Anthony Grant Berry for English correction on earlier draft of the manuscript.

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How to cite this paper: Poolprasert, P., Senarat, S., Nak-eiam, S., Likhitrakarn, N. (2019). Molecular Phylogeny of Predatory Ladybird Beetles (Coleoptera: Coccinellidae) Inferred from COI Sequences. Malaysian Journal of Applied Sciences, 4(2), 10-18.