In the practice of molecular dating, substitution saturation will bias the results if not properly modeled. Date estimates based on commonly used mitochondrial DNA sequences likely suffer from this problem because of their high substitution rate. Nevertheless, the patterns and extent of such expected bias remain unknown for many major evolutionary lineages, which often differ in ages, available calibrations, and substitution rates of their mitochondrial genome. In this case study of salamanders, we used estimates based on multiple nuclear exons to assess the effects of saturation on dating divergences using mitochondrial genome sequences on a timescale of ∼200–300 My. The results indicated that, due to saturation for older divergences and in the absence of younger effective calibration points, dates derived from the mitochondrial data were considerably overestimated and systematically biased toward the calibration point for the ingroup root. The overestimate might be as great as 3–10 times (about 20 My) older than actual divergence dates for recent splitting events and 40 My older for events that are more ancient. For deep divergences, dates estimated were strongly compressed together. Furthermore, excluding the third codon positions of protein-coding genes or only using the RNA genes or second codon positions did not considerably improve the performance. In the order Caudata, slowly evolving markers such as nuclear exons are preferred for dating a phylogeny covering a relatively wide time span. Dates estimated from these markers can be used as secondary calibrations for dating recent events based on rapidly evolving markers for which mitochondrial DNA sequences are attractive candidates due to their short coalescent time. In other groups, similar evaluation should be performed to facilitate the choice of markers for molecular dating and making inferences from the results.

Although mitochondrial (mt) gene order is highly conserved among vertebrates, widespread gene rearrangements occur in anurans, especially in neobatrachians. Protein coding genes in the mitogenome experience adaptive or purifying selection, yet the role that selection plays on genomic reorganization remains unclear. We sequence the mitogenomes of three species of Glandirana and hot spots of gene rearrangements of 20 frog species to investigate the diversity of mitogenomic reorganization in the Neobatrachia. By combing these data with other mitogenomes in GenBank, we evaluate if selective pressures or functional constraints act on mitogenomic reorganization in the Neobatrachia. We also look for correlations between tRNA positions and codon usage. Gene organization in Glandirana was typical of neobatrachian mitogenomes except for the presence of pseudogene trnS (AGY). Surveyed ranids largely exhibited gene arrangements typical of neobatrachian mtDNA although some gene rearrangements occurred. The correlation between codon usage and tRNA positions in neobatrachians was weak, and did not increase after identifying recurrent rearrangements as revealed by basal neobatrachians. Codon usage and tRNA positions were not significantly correlated when considering tRNA gene duplications or losses. Change in number of tRNA gene copies, which was driven by genomic reorganization, did not influence codon usage bias. Nucleotide substitution rates and dN/dS ratios were higher in neobatrachian mitogenomes than in archaeobatrachians, but the rates of mitogenomic reorganization and mt nucleotide diversity were not significantly correlated. No evidence suggests that adaptive selection drove the reorganization of neobatrachian mitogenomes. In contrast, protein-coding genes that function in metabolism showed evidence for purifying selection, and some functional constraints appear to act on the organization of rRNA and tRNA genes. As important nonadaptive forces, genetic drift and mutation pressure may drive the fixation and evolution of mitogenomic reorganizations.

The internal transcribed spacer (ITS) as one part of nuclear ribosomal DNA is one of the most extensively sequenced molecular markers in plant systematics. The ITS repeats generally exhibit high-level within-individual homogeneity, while relatively small-scale polymorphism of ITS copies within individuals has often been reported in literature. Here, we identified large-scale polymorphism of ITS copies within individuals in the legume genus Lespedeza (Fabaceae). Divergent paralogs of ITS sequences, including putative pseudogenes, recombinants, and multiple functional ITS copies were sometimes detected in the same individual. Thirty-seven ITS pseudogenes could be easily detected according to nucleotide changes in conserved 5.8S motives, the significantly lower GC contents in at least one of three regions, and the lost ability of 5.8S rDNA sequence to fold into a conserved secondary structure. The distribution patterns of the putative functional clones were highly different between the traditionally recognized two subgenera, suggesting different rates of concerted evolution in two subgenera which could be attributable to their different extents/frequencies of hybridization, confirmed by our analysis of the single-copy nuclear gene PGK. These findings have significant implications in using ITS marker for reconstructing phylogeny and studying hybridization.

Tandem duplication followed by random loss (TDRL) is the most frequently invoked model to explain the diversity of gene rearrangements in metazoan mitogenomes. The initial stages of gene rearrangement are difficult to observe in nature, which limits our understanding of incipient duplication events and the subsequent process of random loss. Intraspecific gene reorganizations may represent intermediate states, and if so they potentially shed light on the evolutionary dynamics of TDRL. Nucleotide sequences in a hotspot of gene-rearrangement in 28 populations of a single species of frog, Quasipaa boulengeri, provide such predicted intermediate states. Gene order and phylogenetic analyses support a single tandem duplication event and a step-by-step process of random loss. Intraspecific gene rearrangements are not commonly found through comparison of all mitochondrial DNA records of amphibians and squamate reptiles in GenBank. The intraspecific variation in Q. boulengeri provides insights into the rate of partial duplications and deletions within a mitogenome, and reveals that fixation and gene-distribution in mitogenomic reorganization is likely non-adaptive.