Divergence time estimation of genus Tribolium by extensive sampling of highly conserved orthologs.

Tribolium castaneum, the red flour beetle, is among the most well-studied eukaryotic genetic model organisms. Tribolium often serves as a comparative bridge from highly derived Drosophila traits to other organisms. Simultaneously, as a member of the most diverse order of metazoans, Coleoptera, Tribolium informs us about innovations that accompany hyper diversity. However, understanding the tempo and mode of evolutionary innovation requires well-resolved, time-calibrated phylogenies, which are not available for Tribolium. The most recent effort to understand Tribolium phylogenetics used two mitochondrial and three nuclear markers. The study concluded that the genus may be paraphyletic and reported a broad range for divergence time estimates. Here we employ recent advances in Bayesian methods to estimate the relationships and divergence times among Tribolium castaneum, T. brevicornis, T. confusum, T. freemani, and Gnatocerus cornutus using 1368 orthologs conserved across all five species and an independent substitution rate estimate. We find that the most basal split within Tribolium occurred ~86 Mya [95% HPD 85.90-87.04 Mya] and that the most recent split was between T. freemani and T. castaneum at ~14 Mya [95% HPD 13.55-14.00]. Our results are consistent with broader phylogenetic analyses of insects and suggest that Cenozoic climate changes played a role in the Tribolium diversification.

Exploring new dating approaches for parasites: the worldwide Apodanthaceae (Cucurbitales) as an example.

Gene trees of holoparasitic plants usually show distinctly longer branch lengths than seen in photosynthetic closest relatives. Such substitution rate jumps have made it difficult to infer the absolute divergence times of parasites. An additional problem is that parasite clades often lack a fossil record. Using nuclear and mitochondrial DNA sequences of Apodanthaceae, a worldwide family of endoparasites living inside Fabaceae and Salicaceae, we compared several dating approaches: (i) an uncorrelated lognormal (UCLN) model calibrated with outgroup fossils, (ii) ages of host lineages as a maximal age in an UCLN model, (iii) user-assigned local clocks, and (iv) outgroup-fossil-calibrated random local clocks (RLC) with varying prior probabilities on the number of permitted rate changes (RLCu and RLCp models), a variable that has never been explored. The resulting dated phylogenies include all 10 species of the family, three in Australia, one in Iran, one in Africa, and the remainder in the Americas. All clock models infer a drastic rate jump between nonparasitic outgroups and Apodanthaceae, but since they distribute the rate heterogeneity differently, they result in much-different age estimates. Bayes factors using path and stepping-stone sampling indicated that the RLCp model fit poorly, while for matR, topologically unconstrained RLCu and UCLN models did not differ significantly and for 18S, the UCLN model was preferred. Under the equally well fitting models, the Apodanthaceae appear to be a relatively old clade, with a stem age falling between 65 and 81my, the divergence of Apodanthes from Pilostyles between 36 and 57my ago, and the crown age of the Australian clade 8-18my ago. In our study system, host-age calibrations did not yield well-constrained results, but they may work better in other parasite clades. For small data sets where statistical convergence can be reached even with complex models, random local clocks should be explored as an alternative to the exclusive reliance on UCLN clocks.

Estimating Divergence Times and Substitution Rates in Rhizobia.

Accurate estimation of divergence times of soil bacteria that form nitrogen-fixing associations with most leguminous plants is challenging because of a limited fossil record and complexities associated with molecular clocks and phylogenetic diversity of root nodule bacteria, collectively called rhizobia. To overcome the lack of fossil record in bacteria, divergence times of host legumes were used to calibrate molecular clocks and perform phylogenetic analyses in rhizobia. The 16S rRNA gene and intergenic spacer region remain among the favored molecular markers to reconstruct the timescale of rhizobia. We evaluate the performance of the random local clock model and the classical uncorrelated lognormal relaxed clock model, in combination with four tree models (coalescent constant size, birth-death, birth-death incomplete sampling, and Yule processes) on rhizobial divergence time estimates. Bayes factor tests based on the marginal likelihoods estimated from the stepping-stone sampling analyses strongly favored the random local clock model in combination with Yule process. Our results on the divergence time estimation from 16S rRNA gene and intergenic spacer region sequences are compatible with age estimates based on the conserved core genes but significantly older than those obtained from symbiotic genes, such as nodIJ genes. This difference may be due to the accelerated evolutionary rates of symbiotic genes compared to those of other genomic regions not directly implicated in nodulation processes.