Reconstructing species phylogeny of the carabid beetles Ohomopterus using multiple nuclear DNA sequences: heterogeneous information content and the performance of simultaneous analyses.

We attempted a phylogenetic reconstruction for the carabid subgenus Ohomopterus (genus Carabus), a notable case of radiation with mitochondrial introgression across species. Sequence data from five nuclear single copy loci were used, including wingless (Wg), phosphoenolpyruvate carboxykinase (PepCK), cytochrome c (Cytc), elongation factor-1alpha (EF-1alpha), and an anonymous single copy locus (Carab1). Sequences of Cytc, EF-1alpha, and Carab1 included intron or intron-like parts with length variation. The analysis of individual loci resulted in low resolution of the phylogenetic relationships, and the monophyly of several morphologically recognized species for which multiple specimens were analyzed was not revealed. Several specimens were heterozygous, with non-monophyletic alleles observed in three of the five loci at which alleles in heterozygotes were separated. In a simultaneous analysis of the five loci with ambiguously aligned parts eliminated and heterozygotic sites treated as missing, the resulting tree was well resolved, but the branch support was generally weak because of conflicting phylogenetic signals from different loci. We also attempted to incorporate allelic sequence data plus the ambiguously aligned parts in the analysis, by using all possible combinations of alleles from different loci in heterozygotic individuals, but the resultant tree was not supported more strongly. Nonetheless, these simultaneous analyses provided support for the monophyly of several species and species groups, and revealed the basic evolutionary trend of OHOMOPTERUS: initial widespread groups with simpler genitalia and the origination of exaggerated genitalia in a derived clade. This study exemplifies problems inherent in the phylogenetic reconstruction of closely related organisms where low levels of variation limit the information content from each locus, while heterozygosity, different phylogenetic history of multiple loci, and alignment ambiguity further hamper phylogenetic reconstruction unless several loci converge on a uniform signal.