A humanized version of Foxp2 does not affect ultrasonic vocalization in adult mice.

The transcription factor FOXP2 has been linked to severe speech and language impairments in humans. An analysis of the evolution of the FOXP2 gene has identified two amino acid substitutions that became fixed after the split of the human and chimpanzee lineages. Studying the functional consequences of these two substitutions in the endogenous Foxp2 gene of mice showed alterations in dopamine levels, striatal synaptic plasticity, neuronal morphology and cortico-striatal-dependent learning. In addition, ultrasonic vocalizations (USVs) of pups had a significantly lower average pitch than control littermates. To which degree adult USVs would be affected in mice carrying the 'humanized' Foxp2 variant remained unclear. In this study, we analyzed USVs of 68 adult male mice uttered during repeated courtship encounters with different females. Mice carrying the Foxp2(hum/hum) allele did not differ significantly in the number of call elements, their element structure or in their element composition from control littermates. We conclude that neither the structure nor the usage of USVs in adult mice is affected by the two amino acid substitutions that occurred in FOXP2 during human evolution. The reported effect for pup vocalization thus appears to be transient. These results are in line with accumulating evidence that mouse USVs are hardly influenced by vocal learning. Hence, the function and evolution of genes that are necessary, but not sufficient for vocal learning in humans, must be either studied at a different phenotypic level in mice or in other organisms.

Archaeology. The makers of the Protoaurignacian and implications for Neandertal extinction.

The Protoaurignacian culture is pivotal to the debate about the timing of the arrival of modern humans in western Europe and the demise of Neandertals. However, which group is responsible for this culture remains uncertain. We investigated dental remains associated with the Protoaurignacian. The lower deciduous incisor from Riparo Bombrini is modern human, based on its morphology. The upper deciduous incisor from Grotta di Fumane contains ancient mitochondrial DNA of a modern human type. These teeth are the oldest human remains in an Aurignacian-related archaeological context, confirming that by 41,000 calendar years before the present, modern humans bearing Protoaurignacian culture spread into southern Europe. Because the last Neandertals date to 41,030 to 39,260 calendar years before the present, we suggest that the Protoaurignacian triggered the demise of Neandertals in this area.

Complete mitochondrial genomes of ancient canids suggest a European origin of domestic dogs.

The geographic and temporal origins of the domestic dog remain controversial, as genetic data suggest a domestication process in East Asia beginning 15,000 years ago, whereas the oldest doglike fossils are found in Europe and Siberia and date to >30,000 years ago. We analyzed the mitochondrial genomes of 18 prehistoric canids from Eurasia and the New World, along with a comprehensive panel of modern dogs and wolves. The mitochondrial genomes of all modern dogs are phylogenetically most closely related to either ancient or modern canids of Europe. Molecular dating suggests an onset of domestication there 18,800 to 32,100 years ago. These findings imply that domestic dogs are the culmination of a process that initiated with European hunter-gatherers and the canids with whom they interacted.

Targeted resequencing of a genomic region influencing tameness and aggression reveals multiple signals of positive selection.

The identification of the causative genetic variants in quantitative trait loci (QTL) influencing phenotypic traits is challenging, especially in crosses between outbred strains. We have previously identified several QTL influencing tameness and aggression in a cross between two lines of wild-derived, outbred rats (Rattus norvegicus) selected for their behavior towards humans. Here, we use targeted sequence capture and massively parallel sequencing of all genes in the strongest QTL in the founder animals of the cross. We identify many novel sequence variants, several of which are potentially functionally relevant. The QTL contains several regions where either the tame or the aggressive founders contain no sequence variation, and two regions where alternative haplotypes are fixed between the founders. A re-analysis of the QTL signal showed that the causative site is likely to be fixed among the tame founder animals, but that several causative alleles may segregate among the aggressive founder animals. Using a formal test for the detection of positive selection, we find 10 putative positively selected regions, some of which are close to genes known to influence behavior. Together, these results show that the QTL is probably not caused by a single selected site, but may instead represent the joint effects of several sites that were targets of polygenic selection.

Humanized Foxp2 specifically affects cortico-basal ganglia circuits.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution and influence aspects of speech and language. Recently it was shown that when these substitutions are introduced into the endogenous Foxp2 gene of mice, they increase dendrite length and long-term depression (LTD) in medium spiny neurons of the striatum. Here we investigated if these effects are found in other brain regions. We found that neurons in the cerebral cortex, the thalamus and the striatum have increased dendrite lengths in the humanized mice whereas neurons in the amygdala and the cerebellum do not. In agreement with previous work we found increased LTD in medium spiny neurons, but did not detect alterations of synaptic plasticity in Purkinje cells. We conclude that although Foxp2 is expressed in many brain regions and has multiple roles during mammalian development, the evolutionary changes that occurred in the protein in human ancestors specifically affect brain regions that are connected via cortico-basal ganglia circuits.

A locus for an auditory processing deficit and language impairment in an extended pedigree maps to 12p13.31-q14.3.

Despite the apparent robustness of language learning in humans, a large number of children still fail to develop appropriate language skills despite adequate means and opportunity. Most cases of language impairment have a complex etiology, with genetic and environmental influences. In contrast, we describe a three-generation German family who present with an apparently simple segregation of language impairment. Investigations of the family indicate auditory processing difficulties as a core deficit. Affected members performed poorly on a nonword repetition task and present with communication impairments. The brain activation pattern for syllable duration as measured by event-related brain potentials showed clear differences between affected family members and controls, with only affected members displaying a late discrimination negativity. In conjunction with psychoacoustic data showing deficiencies in auditory duration discrimination, the present results indicate increased processing demands in discriminating syllables of different duration. This, we argue, forms the cognitive basis of the observed language impairment in this family. Genome-wide linkage analysis showed a haplotype in the central region of chromosome 12 which reaches the maximum possible logarithm of odds ratio (LOD) score and fully co-segregates with the language impairment, consistent with an autosomal dominant, fully penetrant mode of inheritance. Whole genome analysis yielded no novel inherited copy number variants strengthening the case for a simple inheritance pattern. Several genes in this region of chromosome 12 which are potentially implicated in language impairment did not contain polymorphisms likely to be the causative mutation, which is as yet unknown.

The complex evolutionary history of gorillas: insights from genomic data.

Relatively little is known about the evolutionary and demographic histories of gorillas, one of our closest living relatives. In this study, we used samples from both western (Gorilla gorilla) and eastern (Gorilla beringei) gorillas to infer the timing of the split between these geographically disjunct populations and to elaborate the demographic history of gorillas. Here we present DNA sequences from 16 noncoding autosomal loci from 15 western gorillas and 3 eastern gorillas, including 2 noninvasively sampled free-ranging individuals. We find that the genetic diversity of gorillas is similar to that of chimpanzees but almost twice as high as that of bonobos and humans. A significantly positive Fu & Li's D was observed for western gorillas, suggesting a complex demographic history with a constant, long-term population size and ancestral population structure. Among different population-split scenarios, our data suggest a complex history of western and eastern gorillas including an initial population split at around 0.9-1.6 MYA and subsequent, primarily male-mediated gene flow until approximately 80,000-200,000 years ago. Furthermore, simulations revealed that more gene flow took place from eastern to western gorilla populations than vice versa.

Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA.

Whereas evolutionary inferences derived from present-day DNA sequences are by necessity indirect, ancient DNA sequences provide a direct view of past genetic variants. However, base lesions that accumulate in DNA over time may cause nucleotide misincorporations when ancient DNA sequences are replicated. By repeated amplifications of mitochondrial DNA sequences from a large number of ancient wolf remains, we show that C/G-to-T/A transitions are the predominant type of such misincorporations. Using a massively parallel sequencing method that allows large numbers of single DNA strands to be sequenced, we show that modifications of C, as well as to a lesser extent of G, residues cause such misincorporations. Experiments where oligonucleotides containing modified bases are used as templates in amplification reactions suggest that both of these types of misincorporations can be caused by deamination of the template bases. New DNA sequencing methods in conjunction with knowledge of misincorporation processes have now, in principle, opened the way for the determination of complete genomes from organisms that became extinct during and after the last glaciation.

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Mutations induced by ancient DNA extracts?

We have investigated whether some factor in ancient DNA extracts induces site-specific mutations in modern DNA. We find no evidence for higher mutation rates when extracts from three different Pleistocene mammals are added to modern DNA than when water or extraction blanks are added. We also fail to find evidence that any such factor affects ancient DNA sequences determined from the same extracts. This as well as the patterns of nucleotide substitutions seen in DNA sequences determined from hundreds of other specimens leads us to doubt that a previously unknown mutagenic factor can be a general feature of extracts from old tissues.

Unreliable mtDNA data due to nuclear insertions: a cautionary tale from analysis of humans and other great apes.

Analysis of mitochondrial DNA sequence variation has been used extensively to study the evolutionary relationships of individuals and populations, both within and across species. So ubiquitous and easily acquired are mtDNA data that it has been suggested that such data could serve as a taxonomic 'barcode' for an objective species classification scheme. However, there are technical pitfalls associated with the acquisition of mtDNA data. One problem is the presence of translocated pieces of mtDNA in the nuclear genome of many taxa that may be mistaken for authentic organellar mtDNA. We assessed the extent to which such 'numt' sequences may pose an overlooked problem in analyses of mtDNA from humans and apes. Using long-range polymerase chain reaction (PCR), we generated necessarily authentic mtDNA sequences for comparison with sequences obtained using typical methods for a segment of the mtDNA control region in humans, chimpanzees, bonobos, gorillas and orangutans. Results revealed that gorillas are notable for having such a variety of numt sequences bearing high similarity to authentic mtDNA that any analysis of mtDNA using standard approaches is rendered impossible. Studies on humans, chimpanzees, bonobos or orangutans are apparently less problematic. One implication is that explicit measures need to be taken to authenticate mtDNA sequences in newly studied taxa or when any irregularities arise. Furthermore, some taxa may not be amenable to analysis of mtDNA variation at all.

The genetical history of humans and the great apes.

When and where did modern humans evolve? How did our ancestors spread over the world? Traditionally, answers to questions such as these have been sought in historical, archaeological, and fossil records. However, increasingly genetic data provide information about the evolution of our species. In this review, we focus on the comparison of the variation in the human gene pool to that of our closest evolutionary relatives, the great apes, because this provides a relevant perspective on human genetical evolution. For instance, comparisons to the great apes show that humans are unique in having little genetic variation as well as little genetic structure in their gene pool. Furthermore, genetic data indicate that humans, but not the great apes, have experienced a period of dramatic growth in their early history.

DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.

We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C-->T and G-->A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, 'jumping PCR', i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than approximately 0.1% even under the unlikely scenario where each amplification starts from a single template molecule.

Evidence for import of a lysyl-tRNA into marsupial mitochondria.

The mitochondrial tRNA gene for lysine was analyzed in 11 different marsupial mammals. Whereas its location is conserved when compared with other vertebrate mitochondrial genomes, its primary sequence and inferred secondary structure are highly unusual and variable. For example, eight species lack the expected anticodon. Because the corresponding transcripts are not altered by any RNA-editing mechanism, the lysyl-tRNA gene seems to represent a mitochondrial pseudogene. Purification of marsupial mitochondria and in vitro aminoacylation of isolated tRNAs with lysine, followed by analysis of aminoacylated tRNAs, show that a nuclear-encoded tRNA(Lys) is associated with marsupial mitochondria. We conclude that a functional tRNA(Lys) encoded in the nuclear genome is imported into mitochondria in marsupials. Thus, tRNA import is not restricted to plant, yeast, and protozoan mitochondria but also occurs also in mammals.

Ancient DNA.

DNA that has been recovered from archaeological and palaeontological remains makes it possible to go back in time and study the genetic relationships of extinct organisms to their contemporary relatives. This provides a new perspective on the evolution of organisms and DNA sequences. However, the field is fraught with technical pitfalls and needs stringent criteria to ensure the reliability of results, particularly when human remains are studied.

Molecular analyses of oral polio vaccine samples.

It has been suggested that the human immunodeficiency virus (HIV), and thus the acquired immunodeficiency syndrome (AIDS) it causes, was inadvertently introduced to humans by the use of an oral polio vaccine (OPV) during a vaccination campaign launched by the Wistar Institute, Philadelphia, PA, USA, in the Belgian Congo in 1958 and 1959. The "OPV/AIDS hypothesis" suggests that the OPV used in this campaign was produced in chimpanzee kidney epithelial cell cultures rather than in monkey kidney cell cultures, as stated by H. Koprowski and co-workers, who produced the OPV. If chimpanzee cells were indeed used, this would lend support to the OPV/AIDS hypothesis, since chimpanzees harbor a simian immunodeficiency virus, widely accepted to be the origin of HIV-1. We analyzed several early OPV pools and found no evidence for the presence of chimpanzee DNA; by contrast, monkey DNA is present.

A molecular analysis of dietary diversity for three archaic Native Americans.

DNA was extracted from three fecal samples, more than 2,000 years old, from Hinds Cave, Texas. Amplification of human mtDNA sequences showed their affiliation with contemporary Native Americans, while sequences from pronghorn antelope, bighorn sheep, and cottontail rabbit allowed these animals to be identified as part of the diet of these individuals. Furthermore, amplification of chloroplast DNA sequences identified eight different plants as dietary elements. These archaic humans consumed 2-4 different animal species and 4-8 different plant species during a short time period. The success rate for retrieval of DNA from paleofeces is in strong contrast to that from skeletal remains where the success rate is generally low. Thus, human paleofecal remains represent a source of ancient DNA that significantly complements and may in some cases be superior to that from skeletal tissue.