摘要We present an integrated stand-alone software package named KaKs_Calculator 2.0 as an updated version.It incorporates 17 methods for the calculation of nonsynonymous and synonymous substitution rates; among them,we added our modified versions of several widely used methods as the gamma series including γ=NG,γ-LWL,γ-MLWL,γ-LPB,γ-MLPB,γ-YN and γ-MYN,which have been demonstrated to perform better under certain conditions than their original forms and are not implemented in the previous version.The package is readily used for the identification of positively selected sites based on a sliding window across the sequences of interests in 5'to 3' direction of protein-coding sequences,and have improved the overall performance on sequence analysis for evolution studies.A toolbox,including C++and Java source code and executable files on both Windows and Linux platforms together with a user instruction,is downloadable from the website for academic purpose at https://sourceforge.net/projects/kakscalculator2/.