Michael Hofreiter

Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse

The rich fossil record of equids has made them a model for evolutionary processes. Here we present a 1.12-times coverage draft genome from a horse bone recovered from permafrost dated to approximately 560–780 thousand years before present (kyr bp). Our data represent the oldest full genome sequence determined so far by almost an order of magnitude. For comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr bp), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski’s horse (E. f. przewalskii) and a donkey (E. asinus). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0–4.5 million years before present (Myr bp), twice the conventionally accepted time to the most recent common ancestor of the genus Equus. We also find that horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. We estimate that the Przewalski’s and domestic horse populations diverged 38–72 kyr bp, and find no evidence of recent admixture between the domestic horse breeds and the Przewalski’s horse investigated. This supports the contention that Przewalski’s horses represent the last surviving wild horse population. We find similar levels of genetic variation among Przewalski’s and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. We also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, we identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski’s horse. Such regions could correspond to loci selected early during domestication.

Species-specific responses of Late Quaternary megafauna to climate and humans

Despite decades of research, the roles of climate and humans in driving the dramatic extinctions of large-bodied mammals during the Late Quaternary period remain contentious. Here we use ancient DNA, species distribution models and the human fossil record to elucidate how climate and humans shaped the demographic history of woolly rhinoceros, woolly mammoth, wild horse, reindeer, bison and musk ox. We show that climate has been a major driver of population change over the past 50,000 years. However, each species responds differently to the effects of climatic shifts, habitat redistribution and human encroachment. Although climate change alone can explain the extinction of some species, such as Eurasian musk ox and woolly rhinoceros, a combination of climatic and anthropogenic effects appears to be responsible for the extinction of others, including Eurasian steppe bison and wild horse. We find no genetic signature or any distinctive range dynamics distinguishing extinct from surviving species, emphasizing the challenges associated with predicting future responses of extant mammals to climate and human-mediated habitat change.

Parallel tagged sequencing on the 454 platform

Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplification or gel purification steps. The method relies on attaching sample-specific barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-five 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days.

Ancient DNA extraction from bones and teeth

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20–23 °C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days.

Comparison and optimization of ancient DNA extraction

Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.

Multilocus Resolution of Phylogeny and Timescale in the Extant Adaptive Radiation of Hawaiian Honeycreepers

Evolutionary theory has gained tremendous insight from studies of adaptive radiations. High rates of speciation, morphological divergence, and hybridization, combined with low sequence variability, however, have prevented phylogenetic reconstruction for many radiations. The Hawaiian honeycreepers are an exceptional adaptive radiation, with high phenotypic diversity and speciation that occurred within the geologically constrained setting of the Hawaiian Islands. Here we analyze a new data set of 13 nuclear loci and pyrosequencing of mitochondrial genomes that resolves the Hawaiian honeycreeper phylogeny. We show that they are a sister taxon to Eurasian rosefinches (Carpodacus) and probably came to Hawaii from Asia. We use island ages to calibrate DNA substitution rates, which vary substantially among gene regions, and calculate divergence times, showing that the radiation began roughly when the oldest of the current large Hawaiian Islands (Kauai and Niihau) formed, ~5.7 million years ago (mya). We show that most of the lineages that gave rise to distinctive morphologies diverged after Oahu emerged (4.0-3.7 mya) but before the formation of Maui and adjacent islands (2.4-1.9 mya). Thus, the formation of Oahu, and subsequent cycles of colonization and speciation between Kauai and Oahu, played key roles in generating the morphological diversity of the extant honeycreepers.

Paternity and relatedness in wild chimpanzee communities

The genetic structure of three contiguous wild chimpanzee communities in West Africa was examined to determine the extent to which the community, the mixed-sex social unit of chimpanzees, represents a closed reproductive unit. An analysis of paternity for 41 offspring resulted in 34 cases of paternity assignment to an adult male belonging to the same community. Among the 14 offspring for which all potential within-community fathers have been tested, one likely case of extra-group paternity (EGP) has been identified, suggesting an incidence of EGP of 7%. This more extensive analysis contradicts a previous genetic study of the Taï chimpanzees that inferred 50% extra-group fathers. We suggest, based on direct comparison of results for 33 individuals at 1 microsatellite locus and direct comparison of paternity assignments for 11 offspring, that the error rate in the previous study was too high to produce accurate genotypes and assignments of paternity and hence caused the false inference of a high rate of EGP. Thus, the community is the primary but not exclusive unit for reproduction in wild chimpanzees, and females do not typically reproduce with outside males. Despite the inferred low level of gene flow from extra-community males, relatedness levels among the community males are not significantly higher than among community females, and the distribution of genetic relationships within the group suggests that, rather than a primarily male-bonded social structure, the group is bonded through relationships between males and females. Kinship may explain cooperative behaviors directed against other communities, but is unlikely to explain the high levels of affiliation and cooperation seen for male within-community interactions.

A melanocortin 1 receptor allele suggests varying pigmentation among Neanderthals

The melanocortin 1 receptor (MC1R) regulates pigmentation in humans and other vertebrates. Variants of MC1R with reduced function are associated with pale skin color and red hair in humans of primarily European origin. We amplified and sequenced a fragment of the MC1R gene (mc1r) from two Neanderthal remains. Both specimens have a mutation that was not found in ∼3700 modern humans analyzed. Functional analyses show that this variant reduces MC1R activity to a level that alters hair and/or skin pigmentation in humans. The impaired activity of this variant suggests that Neanderthals varied in pigmentation levels, potentially on the scale observed in modern humans. Our data suggest that inactive MC1R variants evolved independently in both modern humans and Neanderthals.

DNA from Pre-Clovis Human Coprolites in Oregon, North America

The timing of the first human migration into the Americas and its relation to the appearance of the Clovis technological complex in North America at about 11,000 to 10,800 radiocarbon years before the present (14C years B.P.) remains contentious. We establish that humans were present at Paisley 5 Mile Point Caves, in south-central Oregon, by 12,300 14C years B.P., through the recovery of human mitochondrial DNA (mtDNA) from coprolites, directly dated by accelerator mass spectrometry. The mtDNA corresponds to Native American founding haplogroups A2 and B2. The dates of the coprolites are >1000 14C years earlier than currently accepted dates for the Clovis complex.

Targeted high-throughput sequencing of tagged nucleic acid samples

High-throughput 454 DNA sequencing technology allows much faster and more cost-effective sequencing than traditional Sanger sequencing. However, the technology imposes inherent limitations on the number of samples that can be processed in parallel. Here we introduce parallel tagged sequencing (PTS), a simple, inexpensive and flexible barcoding technique that can be used for parallel sequencing any number and type of double-stranded nucleic acid samples. We demonstrate that PTS is particularly powerful for sequencing contiguous DNA fragments such as mtDNA genomes: in theory as many as 250 mammalian mtDNA genomes can be sequenced in a single GS FLX run. PTS dramatically increases the sequencing throughput of samples in parallel and thus fully mobilizes the resources of the 454 technology for targeted sequencing.