Differentiation in stag beetles, Neolucanus swinhoei complex (Coleoptera: Lucanidae): four major lineages caused by periodical Pleistocene glaciations and separation by a mountain range.

Taxonomic debates on Neolucanus swinhoei complex consisting of N. swinhoei, N. doro doro, N. doro horaguchii, and N. euganiae, distributed exclusively in Taiwan, have been ongoing for several decades because of their overlapping morphological characters. To clarify their taxonomic status and phylogeographical history, we analyzed nine morphological characteristics and four molecular amplicons. Phylogenetic inferences based on COI+16S rDNA+wingless showed one eastern and three western lineages, with the latter consisting of one low-hill and two montane lineages. Intermingled DNA sequences from different populations within each lineage, many low FST values, and a high variance component between lineages indicate the possibility of gene flow among populations. However, positive relationships were observed between the genetic divergences of 16S rDNA and its FST values with geographic distance. A divergence estimation based on COI+16S revealed that these beetles might have originated from Asian mainland and differentiated into western and eastern lineages ca. 1Mya, with the differentiation of the western lineages occurring approximately 0.50-0.75Mya. Isolation by mountain ranges and limited flying capability of these beetles as well as populations retreat to and expansion from refugia in response to glaciation cycles have resulted in the current distribution of N. swinhoei complex. Although most morphological characters are variable and undistinguishable, multi-dimensional scaling analysis based on measurable characteristics could recognize hill N. swinhoei as a cluster distinct from the others. However, based on the realities of genetic admixture, shared phylogeographical history and overlapping characteristics, all of these stag beetles should be regarded as Neolucanus swinhoei Bates, 1866.