For over half a century, it has been known that the rate of morphological evolution appears to vary with the time frame of measurement. Rates of microevolutionary change, measured between successive generations, were found to be far higher than rates of macroevolutionary change inferred from the fossil record. More recently, it has been suggested that rates of molecular evolution are also time dependent, with the estimated rate depending on the timescale of measurement. This followed surprising observations that estimates of mutation rates, obtained in studies of pedigrees and laboratory mutation-accumulation lines, exceeded long-term substitution rates by an order of magnitude or more. Although a range of studies have provided evidence for such a pattern, the hypothesis remains relatively contentious. Furthermore, there is ongoing discussion about the factors that can cause molecular rate estimates to be dependent on time. Here we present an overview of our current understanding of time-dependent rates. We provide a summary of the evidence for time-dependent rates in animals, bacteria and viruses. We review the various biological and methodological factors that can cause rates to be time dependent, including the effects of natural selection, calibration errors, model misspecification and other artefacts. We also describe the challenges in calibrating estimates of molecular rates, particularly on the intermediate timescales that are critical for an accurate characterization of time-dependent rates. This has important consequences for the use of molecular-clock methods to estimate timescales of recent evolutionary events.

Recent genomic data has revealed multiple interactions between Neandertals and humans, but there is currently little genetic evidence about Neandertal behavior, diet, or health. We shotgun sequenced ancient DNA from five Neandertal dental calculus specimens to characterize regional differences in Neandertal ecology. At Spy, Belgium, Neandertal diet was heavily meat based, and included woolly rhinoceros and wild sheep-animals characteristic of a steppe environment. In El Sidrón, Spain, no meat was detected in the dental calculus, but dietary components including mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) in Neandertals, suggesting that meat consumption contributed to significant variation between Neandertal microbiota. Evidence for self-medication was identified in one El Sidrón Neandertal with a dental abscess, who also likely suffered from a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Lastly, we characterized a nearly complete genome of the archaeal commensal Methanobrevibacter oralis in Neandertals-the oldest draft microbial genome generated to date at ~48,000 years old (10.2 depth). DNA preserved within dental calculus represents an important new resource of behavioral and health information for ancient hominid specimens, as well as a unique long-term study system for microbial evolution.

The Black Rat (Rattus rattus) spread out of Asia to become one of the world's worst agricultural and urban pests, and a reservoir or vector of numerous zoonotic diseases, including the devastating plague. Despite the global scale and inestimable cost of their impacts on both human livelihoods and natural ecosystems, little is known of the global genetic diversity of Black Rats, the timing and directions of their historical dispersals, and the risks associated with contemporary movements. We surveyed mitochondrial DNA of Black Rats collected across their global range as a first step towards obtaining an historical genetic perspective on this socioeconomically important group of rodents. We found a strong phylogeographic pattern with well-differentiated lineages of Black Rats native to South Asia, the Himalayan region, southern Indochina, and northern Indochina to East Asia, and a diversification that probably commenced in the early Middle Pleistocene. We also identified two other currently recognised species of Rattus as potential derivatives of a paraphyletic R. rattus. Three of the four phylogenetic lineage units within R. rattus show clear genetic signatures of major population expansion in prehistoric times, and the distribution of particular haplogroups mirrors archaeologically and historically documented patterns of human dispersal and trade. Commensalism clearly arose multiple times in R. rattus and in widely separated geographic regions, and this may account for apparent regionalism in their associated pathogens. Our findings represent an important step towards deeper understanding the complex and influential relationship that has developed between Black Rats and humans, and invite a thorough re-examination of host-pathogen associations among Black Rats.

Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR-amplified the blend using five primer sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers.

The evolution of the ratite birds has been widely attributed to vicariant speciation, driven by the Cretaceous breakup of the supercontinent Gondwana. The early isolation of Africa and Madagascar implies that the ostrich and extinct Madagascan elephant birds (Aepyornithidae) should be the oldest ratite lineages. We sequenced the mitochondrial genomes of two elephant birds and performed phylogenetic analyses, which revealed they are the closest relatives of the New Zealand kiwi, and distant from the basal ratite lineage of ostriches. This unexpected result strongly contradicts continental vicariance and instead supports flighted dispersal in all major ratite lineages. We suggest that convergence towards gigantism and flightlessness was facilitated by early Tertiary expansion into the diurnal herbivory niche following the extinction of the dinosaurs.One Sentence Summary: Ancient DNA reveals massive Madagascan elephant birds and tiny NZ kiwi are closest relatives, and supports flighted origin of ratites. Main Text:Despite extensive studies, the evolutionary history of the giant flightless ratite birds of the southern hemisphere landmasses, and the related flighted tinamous of South America, has remained a major unresolved question. The ratites and tinamous, termed palaeognaths due to their shared 'basal' palate structure, form the sister-taxon to all other living birds (neognaths). The living ratites are one of the few bird groups comprised largely of giant terrestrial herbivores and include: the emu and cassowary in Australia and New Guinea; kiwi in New Zealand; ostriches in Africa; and rhea in South America. In addition, two recently extinct groups included the largest birds known: the moa from New Zealand (1) [up to 2-3 m and 250 kg] and elephant birds from Madagascar [2-3 m height but up to 275 kg] (2, 3). Ratites have been believed to have originated through vicariant speciation driven by the continental breakup of Gondwana on the basis of congruence between the sequence of continental rifting and the presumed order of lineage divergence and distribution of ratites (4,5).New Zealand is the only landmass to have supported two major ratite lineages, the giant herbivorous moa and the chicken-sized, nocturnal, omnivorous kiwi. Morphological phylogenetic analyses initially suggested that these two groups were each other's closest relatives (6, 7), presumably diverging after the isolation of an ancestral form following the separation of New Zealand and Australia in the late Cretaceous ca. 80-60 million years ago (Ma) (8). However, subsequent studies suggest that kiwi are more closely related to the Australasian emu and cassowaries (9, 10), while the closest living relatives of the giant moa are the flighted South American tinamous (11-14). The latter relationship was completely unexpected on morphological grounds, and suggests a more complex evolutionary history than predicted by a model of strict vicariant speciation. By rendering ratites paraphyletic, the relationship between moa and tinamous also strong...