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

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 ge