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DNA barcoding and species delimitation of butterflies (Lepidoptera) from Nigeria

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Abstract

Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require both morphological diagnostics and molecular tools, such as DNA barcoding, for correct identification. In particular, the use of the subunit I of the mitochondrial cytochrome c oxidase (COI) gene for DNA barcoding has proven useful in species identification for insects. However, to date, no studies have been carried out on the DNA barcoding of Nigerian butterflies. We evaluated the utility of DNA barcoding applied for the first time to 735 butterfly specimens from southern Nigeria. In total, 699 DNA barcodes, resulting in a record of 116 species belonging to 57 genera, were generated. Our study sample comprised 807 DNA barcodes based on sequences generated from our current study and 108 others retrieved from BOLD. Different molecular analyses, including genetic distance-based evaluation (Neighbor-Joining, Maximum Likelihood and Bayesian trees) and species delimitation tests (TaxonDNA, Automated Barcode Gap Discovery, General Mixed Yule-Coalescent, and Bayesian Poisson Tree Processes) were performed to accurately identify and delineate species. The genetic distance-based analyses resulted in 163 well-separated clusters consisting of 147 described and 16 unidentified species. Our findings indicate that about 90.20% of the butterfly species were explicitly discriminated using DNA barcodes. Also, our field collections reported the first country records of ten butterfly species—Acraea serena, Amauris cf. dannfelti, Aterica galena extensa, Axione tjoane rubescens, Charaxes galleyanus, Papilio lormieri lormeri, Pentila alba, Precis actia, Precis tugela, and Tagiades flesus. Further, DNA barcodes revealed a high mitochondrial intraspecific divergence of more than 3% in Bicyclus vulgaris vulgaris and Colotis evagore. Furthermore, our result revealed an overall high haplotype (gene) diversity (0.9764), suggesting that DNA barcoding can provide information at a population level for Nigerian butterflies. The present study confirms the efficiency of DNA barcoding for identifying butterflies from Nigeria. To gain a better understanding of regional variation in DNA barcodes of this biogeographically complex area, future work should expand the DNA barcode reference library to include all butterfly species from Nigeria as well as surrounding countries. Also, further studies, involving relevant genetic and eco-morphological datasets, are required to understand processes governing mitochondrial intraspecific divergences reported in some species complexes.

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Acknowledgements

We are grateful to field assistants at the Cross River National Park Nigeria, for their help during sampling. The authors wish to thank Deme Gideon Gyma and Daniel Bassey for their assistance during the field survey.

Funding

This work was supported by the Whitley Wildlife Conservation Trust, Sino-Africa Joint Research Centre, Chinese Academy of Sciences (SAJC201611), National Natural Science Foundation of China (31750110480), and the Animal Branch of the Germplasm Bank of Wild Species, Chinese Academy of Sciences (the Large Research Infrastructure Funding).

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  1. Lotanna Micah Nneji and Adeniyi Charles Adeola have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, China

    Lotanna Micah Nneji, Adeniyi Charles Adeola, Adeola Oluwakemi Ayoola, Yun-Yu Wang & Md Mizanur Rahman

  2. Sino-Africa Joint Research Centre, Chinese Academy of Sciences, Kunming, 650223, Yunnan, China

    Lotanna Micah Nneji & Adeniyi Charles Adeola

  3. Department of Biosciences and Biotechnology, Kwara State University, Malete, Kwara, Nigeria

    Segun Olayinka Oladipo

  4. Department of Biological Science, University of Abuja, Abuja, 902101, Federal Capital Territory, Nigeria

    Yoila D. Malann & Ifeanyi Christopher Nneji

  5. Department of Zoology, Faculty of Science, University of Ibadan, Ibadan, 200, Oyo, Nigeria

    Okorie Anyaele

  6. Cross River National Park, Calabar, 542, Cross River, Nigeria

    Caroline Samuel Olory

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  1. Lotanna Micah Nneji
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Contributions

LMN and ACA designed and supervised the project. LMN, AOA, OA, YM., SOO, ICN, and AOA collected the samples. LMN, ACA, and YYW performed the molecular laboratory works. LMN and MMR performed genetic analyses. CSO provided technical assistance. LMN wrote the initial draft of the manuscript. ACA revised the initial draft of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Lotanna Micah Nneji or Adeniyi Charles Adeola.

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All authors have declare that no conflicts of interest exist.

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Permission to collect animal specimens (NPH/GEN/121/XXV/461) from Cross River National Park was obtained from the National Park Service Headquarter, Abuja, Nigeria.

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Electronic supplementary material

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Electronic supplementary material 1 (DOCX 100 kb)

Electronic supplementary material 2 (DOCX 26 kb)

Electronic supplementary material 3 (DOCX 16 kb)

Electronic supplementary material 4 (DOCX 31 kb)

Electronic supplementary material 5 (DOCX 34 kb)

11033_2020_5984_MOESM6_ESM.pdf

Electronic supplementary material 6 (PDF 4196 kb). Figure S1: The Neighbor-Joining tree used in the identification of the newly collected butterfly species from Cross River National Park, Nigeria, Note = queried sequences are highlighted in red. The Neighbor-Joining trees were constructed using BOLD web interphase

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Electronic supplementary material 7 (PDF 1469 kb). Figure S2: Bootstrap neighbour-joining tree of all 807 individuals of butterflies from Nigeria used in the analysis, estimated on the basis of Kimura 2-parameter

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Electronic supplementary material 8 (PDF 381 kb). Figure S3: The Maximum likelihood (ML) tree based on the GTR + G model and obtained using the 304 unique COI haplotypes of Nigerian butterflies. Numbers above or below branches correspond to bootstrap support based on 1,000 pseudoreplicates

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Electronic supplementary material 9 (PDF 352 kb). The Bayesian Inference tree based on the 304 unique COI haplotypes of Nigerian butterflies

11033_2020_5984_MOESM10_ESM.pdf

Electronic supplementary material 10 (PDF 631 kb). (A) The initial and (b) recursive partitions of the ABGD tree-based identification based on Simple distance method using the 304 unique COI haplotypes of Nigerian butterflies

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Electronic supplementary material 11 (PDF 655 kb). (A) The initial and (b) recursive partitions of the ABGD tree-based identification based on JC method using the 304 unique COI haplotypes of Nigerian butterflies

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Electronic supplementary material 12 (PDF 547 kb). (A) The initial and (b) recursive partitions of the ABGD tree-based identification based on Kimura method using the 304 unique COI haplotypes of Nigerian butterflies

11033_2020_5984_MOESM13_ESM.pdf

Electronic supplementary material 13 (PDF 595 kb). The bPTP tree-based identification based on (A) Bayesian Inference and (2) Maximum Likelihood trees using the 304 unique COI haplotypes of Nigerian butterflies

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Nneji, L.M., Adeola, A.C., Ayoola, A.O. et al. DNA barcoding and species delimitation of butterflies (Lepidoptera) from Nigeria. Mol Biol Rep 47, 9441–9457 (2020). https://doi.org/10.1007/s11033-020-05984-5

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