Proof on the Divergence Times of Two Sympatric Species, Rhynchophorus ferrugineus and R. vulneratus (Coleoptera: Curculionidae) by Molecular Clock Analysis

Abstract

Molecular clock analysis has clearly separated Rhynchophorus ferrugineus and Rhynchophorus vulneratus as two sympatric species. Calibration was performed using the fossil of R. cruentatus which evolved approximately 1 mya using sequences data of COI and Cytb combined. Divergence time indicated that R. cruentatus, the outgroup species evolved around 80.598±10 mya, followed by R. vulneratus (≈29.861±10 mya), and finally speciated to form R. ferrugineus (≈1.926±10 mya) and R. vulneratus (≈4.857±10 mya). Additionally, construction of NJ and MP trees showed distinct separation using COI, Cytb and combination of COI and Cytb. These findings were highly supported by genetic distance analysis. Although a limited number of individuals from small geographical area were used in this study, the genetic molecular clock analysis of mitochondrial data was able to effectively differentiate both species. These results reveal the first analysis to use the molecular clock to confirm separation of these two sympatric species.