Many questions in evolutionary biology require an estimate of divergence times but, for groups with a sparse fossil record, such estimates rely heavily on molecular dating methods. The accuracy of these methods depends on both an adequate underlying model and the appropriate implementation of fossil evidence as calibration points. We explore the effect of these in Poaceae (grasses), a diverse plant lineage with a very limited fossil record, focusing particularly on dating the early divergences in the group. We show that molecular dating based on a dataset of plastid markers is strongly dependent on the model assumptions. In particular, an acceleration of evolutionary rates at the base of Poaceae followed by a deceleration in the descendants strongly biases methods that assume an autocorrelation of rates. This problem can be circumvented by using markers that have lower rate variation, and we show that phylogenetic markers extracted from complete nuclear genomes can be a useful complement to the more commonly used plastid markers. However, estimates of divergence times remain strongly affected by different implementations of fossil calibration points. Analyses calibrated with only macrofossils lead to estimates for the age of core Poaceae around 51-55 Ma, but the inclusion of microfossil evidence pushes this age to 74-82 Ma and leads to lower estimated evolutionary rates in grasses. These results emphasize the importance of considering markers from multiple genomes and alternative fossil placements when addressing evolutionary issues that depend on ages estimated for important groups.
genomes
Data set of nuclear genes used for dating analyses
genomes_beast_1
This file contains the consensus tree inferred on nuclear markers by BEAST with the topology fixed. External calibration only was used. Each time unit corresponds to 20 million years.
genomes_beast_2
This file contains the consensus tree inferred on nuclear markers by BEAST with the topology fixed. External calibration plus phytoliths were used. Each time unit corresponds to 20 million years.
plastid28
Data set of plastid markers for 28 species used for dating analyses.
plastid245
Data set of plastid markers for 245 species used for dating analyses.
plastid245_beast_1
This file contains the consensus tree inferred on plastid markers by BEAST with the topology fixed. External calibration only was used. Each time unit corresponds to 20 million years.
plastid245_beast_2
This file contains the consensus tree inferred on plastid markers by BEAST with the topology fixed. External calibration plus phytoliths were used. Each time unit corresponds to 20 million years.
FigureS1
Supplementary Figure 1: Comparison of concatenated and partitioned analyses. Ages are plotted in million years ago for a) plastid markers and b) nuclear markers. Analyses were performed with BEAST and the dataset was partitioned by codon position. Black lines indicate 1:1 relationships.
FigureS2
Supplementary Figure 2: Comparison of prior and posterior distribution of nodes for the plastid phylogeny under calibration #1. For each calibration point, the calibration density is indicated by the black line, the marginal prior distribution by the blue bars and the posterior distribution by the red bars.
FigureS3
Supplementary Figure 3: Comparison of prior and posterior distribution of nodes for the plastid phylogeny under calibration #2. For each calibration point, the calibration density is indicated by the black line, the marginal prior distribution by the blue bars and the posterior distribution by the red bars.
FigureS4
Supplementary Figure 4: Comparison of prior and posterior distribution of nodes for the nuclear phylogeny under calibration #1. For each calibration point, the calibration density is indicated by the black line, the marginal prior distribution by the blue bars and the posterior distribution by the red bars.
FigureS5
Supplementary Figure 5: Comparison of prior and posterior distribution of nodes for the nuclear phylogeny under calibration #2. For each calibration point, the calibration density is indicated by the black line, the marginal prior distribution by the blue bars and the posterior distribution by the red bars.
FigureS6
Supplementary Figure 6: Comparison of evolutionary rates. Boxplots of inferred evolutionary rates (in expected mutations per site per billion years) are shown for plastid markers (top row) and nuclear markers (bottom row) under calibration #1. Rates are sorted by phylogenetic groups: basal angiosperms (“basal”), eudicots, monocots without graminids, graminids without BEP-PACMAD and BEP-PACMAD. The results obtained with four different methods are shown: BEAST, PB_ug = uncorrelated gamma method implemented in PHYLOBAYES, PB_ln = log-normal autocorrelated method implemented in PHYLOBAYES, MD = MULTIDIVTIME.
FigureS7
Supplementary Figure 7: Age for the BEP-PACMAD crown estimated by MULTIDIVTIME under different priors. The age for the node (in million years ago) is indicated as a function of the prior for the mean of the Brownian motion constant mean. Results are aggregated for different values of the other priors. Analyses with a value of 0.01 for the Brownian motion constant standard deviation are in black.
FigureS8
Supplementary Figure 8: Detailed comparison of methods and datasets. For calibration #1, the age estimates (in million years ago) are represented for nodes that were shared between phylogenetic trees of plastid and nuclear markers. The ages are represented independently when produced by BEAST and MULTIDIVTIME. Black circles are ages based on the whole plastid dataset, red squares are ages based on plastid markers for 28 species only, black triangles are aged based on the whole nuclear dataset, and red bars indicate the interval between the 1st and 3rd quartiles of 100 replicates of the nuclear dataset decreased to the size of the plastid dataset. Taxonomic groups are indicated on the bottom. The last point corresponds to the crown of BEP, and the horizontal bar indicates the minimal age for the clade that would be congruent with the 67 Ma phytolith fossil (Prasad et al. 2011). Numbers can be used to identify the corresponding nodes in Supplementary Figure 9.
FigureS9
Supplementary Figure 9: Node identity. Nodes represented in Figure 2 and Supplementary Figure 8 are indicated on the phylogenetic tree showing the expected relationships among nuclear genes without branch lengths. Taxonomic groups represented in the tips are delimited on the right.