Exploring Patterns and Extent of Bias in Estimating Divergence Time from Mitochondrial DNA Sequence Data in a Particular Lineage: A Case Study of Salamanders (Order Caudata)

Abstract

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.

What explains patterns of species richness? The relative importance of climatic‐niche evolution, morphological evolution, and ecological limits in salamanders

“…However, this tree gives similar ages to those based on multiple nuclear loci analyzed with the Bayesian uncorrelated lognormal approach (Zheng et al. 2011) for major clades within Plethodontidae (for specific comparisons see Fisher‐Reid et al. 2012).…”

Phylogenetic relationships of Amazonian anole lizards (Dactyloa): Taxonomic implications, new insights about phenotypic evolution and the timing of diversification

“…For instance, the phylogenetic placement of the semi-fossils used for node calibration has been uncertain (Castañeda et al, 2014;de Queiroz et al, 1998;Lazell, 1965). Moreover, the divergence time estimates presented by Nicholson et al (2012) were based on a single mitochondrial marker, yet mitochondrial genes are known to mislead deep divergence time estimation due to substitution saturation (Brandley et al, 2011;Lukoschek et al, 2012;Mulcahy et al, 2012), sometimes resulting in 3-10-fold overestimations (Zheng et al, 2011). To improve estimates of divergence times for Amazonian anoles, we built a dated phylogeny for the pleurodont iguanian clade (sensu Wiens et al, 2012) using five nuclear genes and well-known fossils for calibration points.…”

A reinvestigation of phylogeny and divergence times of Hynobiidae (Amphibia, Caudata) based on 29 nuclear genes

“…However, another analysis also based on mitochondrial genomes with an improved taxon sampling modified the position of Pachyhynobius, although not conclusively (Peng et al, 2010). More recently, Zheng et al (2011) used mtDNA genome data and three nuclear genes to investigate phylogeny and divergence times for Hynobiidae, including 25 hynobiid species with representatives of all genera. In that study, the phylogenetic tree inferred from mitochondrial genomes was nearly identical to that of Zhang et al (2006), whereas different placements of Pachyhynobius and Salamandrella were obtained using nuclear genes.…”

“…In addition, previous dating analyses for hynobiid evolution using mitochondrial genomes (Zhang et al, 2006) or a few nuclear genes (Zheng et al, 2011) have also yielded conflicting results. Times derived from the mitochondrial data are much older than those estimated from nuclear data when the reference point for calibration is a fossil-based estimate of an event preceding the phylogenetic divergences being studied.…”

“…Times derived from the mitochondrial data are much older than those estimated from nuclear data when the reference point for calibration is a fossil-based estimate of an event preceding the phylogenetic divergences being studied. Recent empirical studies have consistently shown that slowly evolving markers such as nuclear exons surpass mitochondrial data for dating divergences exceeding 15 Mya (Zheng et al, 2011;Near et al, 2012). However, because nuclear genes are relatively conservative and contain fewer signal sites, more loci are needed to calculate reliable divergence times.…”