Abstract
Molecular clocks have become powerful tools given increasing sequencing and fossil resources. However, calibration analyses outcomes depend on the choice of priors. Here, we revisited the seminal dating study published by Andújar and coworkers of the genus Carabus proposing that prior choices need re-evaluation. We hypothesized that reflecting fossil evidence and the Gondwanan split properly significantly rewinds the molecular clock. We re-used the dataset including five mitochondrial and four nuclear DNA fragments with a total length of 7888 nt. Fossil evidence for Oligocene occurrence of Calosoma was considered. Root age was set based on the fossil evidence of Harpalinae ground beetles in the Upper Cretaceous. Paleogene divergence of the outgroup taxa Ceroglossini and Pamborini is introduced as a new prior based on current paleontological and geological literature. The ultrametric time-calibrated tree of the extended nd5 dataset resulted in a median TMRCA Carabus of 53.92 Ma (HPD 95% 45.01–63.18 Ma), roughly 30 Ma older than in the Andújar study. The splits among C. rugosus and C. morbillosus (A), C. riffensis from the European Mesocarabus (B), and Eurycarabus and Nesaeocarabus (C) were dated to 17.58 (12.87–22.85), 24.14 (18.02–30.58), and 21.6 (16.44–27.43) Ma. They were decidedly older than those previously reported (7.48, 10.93, and 9.51 Ma). These changes were driven almost entirely by constraining the Carabidae time-tree root with a Harpalinae amber fossil at ~99 Ma. Utilizing the nd5 dating results of three well-supported Carabus clades as secondary calibration points for the complete MIT-NUC dataset led to a TMRCA of Carabus of 44.72 (37.54–52.22) Ma, compared with 25.16 Ma (18.41–33.04 Ma) in the previous study. Considering fossil evidence for Oligocene Calosoma and Late Cretaceous Harpalini together with the Gondwanan split as a new prior, our new approach supports the origin of genus Carabus in the Eocene. Our results are preliminary because of the heavy reliance on the nd5 gene, and thus will have to be tested with a sufficient set of nuclear markers. Additionally, uncertainties due to dating root age of the tree based on a single fossil and outgroup taxon affect the results. Improvement of the fossil database, particularly in the supertribe Carabitae, is needed to reduce these uncertainties in dating Carabus phylogeny.
Highlights
The molecular clock has become an increasingly powerful tool in biogeography and phylogenetics owing to ever-increasing genomic and fossil calibration data [1]
Since Andujar et al [16] neglected this calibration point, we propose that their time-tree massively underestimates the true age of the genus Carabus, as was already presumed by Toussaint & Gillet [25]
Our findings of the Carabus time-tree are used to discuss the general need for a more differentiated and transparent usage of geological calibration points depending on life-history traits and habitat requirements of the taxa under study
Summary
The molecular clock has become an increasingly powerful tool in biogeography and phylogenetics owing to ever-increasing genomic and fossil calibration data [1]. There is often a huge dating variance among studies, even those dealing with identical taxa and employing identical calibration points [3,4,5]. Important factors for these variances are the placement of fossils in a given phylogeny and the handling of geological priors. Recent methodological improvements for better analyses of hidden characters in fossils have included X-ray micro-computed tomography of amber inclusions to determine internal genital characteristics of tiny beetles [6, 7]. The geological record should be used to calibrate the molecular clock only for taxa which are poor dispersers
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