Abstract: 【Aim】This study aims to explore the genetic diversity, genetic differentiation, and phylogenetic relationships among populations of the pierid butterfly Colias fieldii in China, to infer their origin and divergence time, and to preliminarily clarify the causes of their spatiotemporally evolutionary history. 【Methods】 The four mitochondrial gene (COI, Cytb, NDI and ND5) sequences of 115 individuals from 23 geographic populations of C. fieldii in China collected in 2006-2018 were amplified by PCR and sequenced. The genetic diversity and genetic differentiation were analyzed using MEGA v.7.0, DnaSP v.5.0, Arlequin v.3.5.1 and other genetic analysis software. Using the closest relatives as the outgroups, the phylogenetic trees and haplotype median-joining network of C. fieldii were reconstructed with such analytical software as IQ-TREE, MrBayes v.3.1.2, Network v.4.6 and BEAST v.1.8.3, and the origin and divergence time of C. fieldii were estimated by using relaxed molecular dating method and calibrations of the previous studies. Based on the present biogeographic distribution of C. fieldii and the main earth environmental events since the Quaternary Period, the spatio-temporal pattern of its biogeographic distribution and the underlying earth environmental factors were tentatively inferred. 【Results】 The aligned sizes of mitochondrial gene segments of COI, Cytb, NDI and ND5 of C. fieldii populations are 648, 699, 393 and 777 bp, respectively, and the concatenated sequence of the four genes is 2 517 bp in length, which was shown to be significantly AT biased. In total, 18 haplotypes based on the four mitochondrial gene sequences were found in 115 individuals of 23 geographic populations of C. fieldii, with the haplotype diversity (Hd) of 0.677±0.048 and nucleotide diversity (π) of 0.00066±0.00007 of the total population, showing a relatively high level of haplotype diversity and a low level of nucleotide diversity. The phylogenetic analysis showed that 18 haplotypes of C. fieldii populations were distinctly categorized into two large clades (clade I and II). Clade I included 13 haplotypes of populations from Shaanxi, Henan, Gansu, Anhui, Hubei, Sichuan, Qinghai, and some regions of Yunnan. Clade II included five haplotypes of populations from some regions of Yunnan and Tibet. The results of reconstructed haplotype median-joining network were generally consistent with those of the phylogenetic tree. AMOVA analysis indicated that a larger level of population differentiation (64.36%) occurred between the two haplotype clades and a subtle genetic differentiation (35.64%) existed within each haplotype clade of C. fieldii. The analysis results of neutrality tests and mismatch distribution indicated that populations with haplotypes in clade I did not experience a population expansion event, whereas those with haplotypes in clade II probably had a sudden demographic expansion at about 0.085 Ma in the late Pleistocene, a little earlier than the Last Glacial Maximum event, which may be caused by the warm and humid plateau climate in the interglacial period, the expansion of forest grassland and the decreased heavy rainfall on the core plateau. 【Conclusion】 The genetic differentiation of C. fieldii populations is correlated significantly with the geographical distance. In addition, we proposed that C. fieldii populations originated at about 0.48 Ma in southwestern areas of China (presently the Hengduan Mountains and adjacent areas), and began to diversify into two clades and later dispersed into other low-latitude areas due to the Quaternary glacial-interglacial cycle events, Southeast Asia monsoon and different habitat environments.

Key words:  Colias fieldii, genetic differentiation, haplotype, mitochondrial gene, molecular dating, phylogeography, China