DOI: 10.5061/dryad.1zcrjdfp5
title: Quantifying the error of secondary vs. distant primary calibrations in a simulated environment

description: Using calibrations to obtain absolute divergence times is standard
 practice in molecular clock studies. While the use of primary (e.g.,
 fossil) calibrations is preferred, this approach can be limiting because
 of their rarity in fast-growing datasets. Thus, alternatives need to be
 explored, such as the use of secondary (molecularly-derived) calibrations
 that can anchor a timetree in a larger number of nodes. However, the use
 of secondary calibrations has been discouraged in the past because of
 concerns in the error rates of the node estimates they produce with an
 apparent high precision. Here, we quantify the amount of errors in
 estimates produced by the use of secondary calibrations relative to true
 times and primary calibrations placed on distant nodes. We find that,
 overall, the inaccuracies in estimates based on secondary calibrations are
 predictable and mirror errors associated with primary calibrations and
 their confidence intervals. Additionally, we find comparable error rates
 in estimated times from secondary calibrations and distant primary
 calibrations, although the precision of estimates derived from distant
 primary calibrations is roughly twice as good as that of estimates derived
 from secondary calibrations. This suggests that increasing dataset size to
 include primary calibration may produce divergence times that are about as
 accurate as those from secondary calibrations, albeit with a higher
 precision. Overall, our results suggest that secondary calibrations may be
 useful to explore the parameter space of plausible evolutionary scenarios
 when compared to time estimates obtained with distant primary
 calibrations.
description: We started from a main tree
 of 248 species represented in a tree of life. This main tree was split
 into two subtrees, tree A (173 species) and tree B (71 species), that
 represent two clades and maximize the size of the dataset in each tree. We
 then added to these clades two shared lineages which were arbitrarily
 chosen and an outgroup. This setup created two nested phylogenies that
 were used to test hypotheses on calibrations’ performance. To simulate
 multiple genes, we used a set of 446 empirical parameters (e.g., length,
 GC content, initial evolutionary rate) and altered the main timetree
 according to an autocorrelated model (ν = 1) that resulted in estimated
 rates of up to ± 25% of the mean rate. This effectively created 446
 phylogenies with different branch lengths but same topology. These
 parameters were given to SeqGen to simulate genes under an
 Hasegawa-Kishino-Yano (HKY) model. Ten random sets of individual genes
 were then concatenated to reach a length of at least 30,000 sites (30,029
 – 30,725). In addition, we also created one concatenation with all genes
 (approximately 604,000 sites) and two concatenations of half the number of
 genes (223 genes per concatenation) with lengths of 273,812 and 330,187.
 Each of these concatenations were used independently in downstream
 analyses. Patterns between the 30k, half, and full concatenations were
 similar. Therefore, we discuss results from the 30k concatenations because
 they allow us to evaluate the variance of estimates among datasets. For
 primary calibrations, three nodes from tree A were chosen: a relatively
 shallow node at 63.9 million years ago (mya), and two that were deeper in
 the tree but in two different clades (209.4 mya and 220.2 mya). The
 overlapping node between tree A and B has an intermediate depth (167 mya)
 within tree A and is centrally placed within the topology of tree B. These
 primary and secondary calibrations were chosen to minimize the effect that
 biased location (e.g., all within one clade) and divergence times (e.g.,
 all young nodes) may have on the accuracy of estimations.
description: This document contains simulated sequences used to estimate
 divergence times with primary and secondary calibrations.