Ancient and Modern Genomes Reveal Microsatellites Maintain a Dynamic Equilibrium Through Deep Time.

Microsatellites are widely used in population genetics, but their evolutionary dynamics remain poorly understood. It is unclear whether microsatellite loci drift in length over time. This is important because the mutation processes that underlie these important genetic markers are central to the evolutionary models that employ microsatellites. We identify more than 27 million microsatellites using a novel and unique dataset of modern and ancient Adélie penguin genomes along with data from 63 published chordate genomes. We investigate microsatellite evolutionary dynamics over 2 timescales: one based on Adélie penguin samples dating to ∼46.5 ka and the other dating to the diversification of chordates aged more than 500 Ma. We show that the process of microsatellite allele length evolution is at dynamic equilibrium; while there is length polymorphism among individuals, the length distribution for a given locus remains stable. Many microsatellites persist over very long timescales, particularly in exons and regulatory sequences. These often retain length variability, suggesting that they may play a role in maintaining phenotypic variation within populations.

Ancient nuclear genomes enable repatriation of Indigenous human remains.

After European colonization, the ancestral remains of Indigenous people were often collected for scientific research or display in museum collections. For many decades, Indigenous people, including Native Americans and Aboriginal Australians, have fought for their return. However, many of these remains have no recorded provenance, making their repatriation very difficult or impossible. To determine whether DNA-based methods could resolve this important problem, we sequenced 10 nuclear genomes and 27 mitogenomes from ancient pre-European Aboriginal Australians (up to 1540 years before the present) of known provenance and compared them to 100 high-coverage contemporary Aboriginal Australian genomes, also of known provenance. We report substantial ancient population structure showing strong genetic affinities between ancient and contemporary Aboriginal Australian individuals from the same geographic location. Our findings demonstrate the feasibility of successfully identifying the origins of unprovenanced ancestral remains using genomic methods.

The Sacred Ibis debate: The first test of evolution.

In 1798, Napoleon Bonaparte's army invaded Egypt, returning with many treasures including large numbers of Sacred Ibis mummies. The ancient Egyptians revered the ibis and mummified literally millions of them. The French naturalist Georges Cuvier used these mummies to challenge an emerging idea of the time, namely Jean-Baptiste Lamarck's theory of evolution. Cuvier detected no measurable differences between mummified Sacred Ibis and contemporary specimens of the same species. Consequently, he argued that this was evidence for the "fixity of species." The "Sacred Ibis debate" predates the so-called "Great Debate" between Cuvier and Geoffroy Saint-Hilaire and the publication of Darwin's On the Origin of Species five decades later. Cuvier's views and his study had a profound influence on the scientific and public perception of evolution, setting back the acceptance of evolutionary theory in Europe for decades.

Co-option of the cardiac transcription factor Nkx2.5 during development of the emu wing.

The ratites are a distinctive clade of flightless birds, typified by the emu and ostrich that have acquired a range of unique anatomical characteristics since diverging from basal Aves at least 100 million years ago. The emu possesses a vestigial wing with a single digit and greatly reduced forelimb musculature. However, the embryological basis of wing reduction and other anatomical changes associated with loss of flight are unclear. Here we report a previously unknown co-option of the cardiac transcription factor Nkx2.5 to the forelimb in the emu embryo, but not in ostrich, or chicken and zebra finch, which have fully developed wings. Nkx2.5 is expressed in emu limb bud mesenchyme and maturing wing muscle, and mis-expression of Nkx2.5 throughout the limb bud in chick results in wing reductions. We propose that Nkx2.5 functions to inhibit early limb bud expansion and later muscle growth during development of the vestigial emu wing.The transcription factor Nkx2.5 is essential for heart development. Here, the authors identify a previously unknown expression domain for Nkx2.5 in the emu wing and explore its role in diminished wing bud development in the flightless emu, compared with three other birds that have functional wings.

Limb patterning genes and heterochronic development of the emu wing bud.

BACKGROUND: The forelimb of the flightless emu is a vestigial structure, with greatly reduced wing elements and digit loss. To explore the molecular and cellular mechanisms associated with the evolution of vestigial wings and loss of flight in the emu, key limb patterning genes were examined in developing embryos. METHODS: Limb development was compared in emu versus chicken embryos. Immunostaining for cell proliferation markers was used to analyze growth of the emu forelimb and hindlimb buds. Expression patterns of limb patterning genes were studied, using whole-mount in situ hybridization (for mRNA localization) and RNA-seq (for mRNA expression levels). RESULTS: The forelimb of the emu embryo showed heterochronic development compared to that in the chicken, with the forelimb bud being retarded in its development. Early outgrowth of the emu forelimb bud is characterized by a lower level of cell proliferation compared the hindlimb bud, as assessed by PH3 immunostaining. In contrast, there were no obvious differences in apoptosis in forelimb versus hindlimb buds (cleaved caspase 3 staining). Most key patterning genes were expressed in emu forelimb buds similarly to that observed in the chicken, but with smaller expression domains. However, expression of Sonic Hedgehog (Shh) mRNA, which is central to anterior-posterior axis development, was delayed in the emu forelimb bud relative to other patterning genes. Regulators of Shh expression, Gli3 and HoxD13, also showed altered expression levels in the emu forelimb bud. CONCLUSIONS: These data reveal heterochronic but otherwise normal expression of most patterning genes in the emu vestigial forelimb. Delayed Shh expression may be related to the small and vestigial structure of the emu forelimb bud. However, the genetic mechanism driving retarded emu wing development is likely to rest within the forelimb field of the lateral plate mesoderm, predating the expression of patterning genes.

Genomic analyses inform on migration events during the peopling of Eurasia.

High-coverage whole-genome sequence studies have so far focused on a limited number of geographically restricted populations, or been targeted at specific diseases, such as cancer. Nevertheless, the availability of high-resolution genomic data has led to the development of new methodologies for inferring population history and refuelled the debate on the mutation rate in humans. Here we present the Estonian Biocentre Human Genome Diversity Panel (EGDP), a dataset of 483 high-coverage human genomes from 148 populations worldwide, including 379 new genomes from 125 populations, which we group into diversity and selection sets. We analyse this dataset to refine estimates of continent-wide patterns of heterozygosity, long- and short-distance gene flow, archaic admixture, and changes in effective population size through time as well as for signals of positive or balancing selection. We find a genetic signature in present-day Papuans that suggests that at least 2% of their genome originates from an early and largely extinct expansion of anatomically modern humans (AMHs) out of Africa. Together with evidence from the western Asian fossil record, and admixture between AMHs and Neanderthals predating the main Eurasian expansion, our results contribute to the mounting evidence for the presence of AMHs out of Africa earlier than 75,000 years ago.

Ancient mtDNA sequences from the First Australians revisited.

The publication in 2001 by Adcock et al. [Adcock GJ, et al. (2001) Proc Natl Acad Sci USA 98(2):537-542] in PNAS reported the recovery of short mtDNA sequences from ancient Australians, including the 42,000-y-old Mungo Man [Willandra Lakes Hominid (WLH3)]. This landmark study in human ancient DNA suggested that an early modern human mitochondrial lineage emerged in Asia and that the theory of modern human origins could no longer be considered solely through the lens of the "Out of Africa" model. To evaluate these claims, we used second generation DNA sequencing and capture methods as well as PCR-based and single-primer extension (SPEX) approaches to reexamine the same four Willandra Lakes and Kow Swamp 8 (KS8) remains studied in the work by Adcock et al. Two of the remains sampled contained no identifiable human DNA (WLH15 and WLH55), whereas the Mungo Man (WLH3) sample contained no Aboriginal Australian DNA. KS8 reveals human mitochondrial sequences that differ from the previously inferred sequence. Instead, we recover a total of five modern European contaminants from Mungo Man (WLH3). We show that the remaining sample (WLH4) contains ∼1.4% human DNA, from which we assembled two complete mitochondrial genomes. One of these was a previously unidentified Aboriginal Australian haplotype belonging to haplogroup S2 that we sequenced to a high coverage. The other was a contaminating modern European mitochondrial haplotype. Although none of the sequences that we recovered matched those reported by Adcock et al., except a contaminant, these findings show the feasibility of obtaining important information from ancient Aboriginal Australian remains.

A Concentrated Hydrochloric Acid-based Method for Complete Recovery of DNA from Bone.

The successful extraction of DNA from historical or ancient animal bone is important for the analysis of discriminating genetic markers. Methods used currently rely on the digestion of bone with EDTA and proteinase K, followed by purification with phenol/chloroform and silica bed binding. We have developed a simple concentrated hydrochloric acid-based method that precludes the use of phenol/chloroform purification and can lead to a several-fold increase in DNA yield when compared to other commonly used methods. Concentrated hydrochloric acid was shown to dissolve most of the undigested bone and allowed the efficient recovery of DNA fragments <100 bases in length. This method should prove useful for the recovery of DNAs from highly degraded animal bone, such as that found in historical or ancient samples.

Distance-dependent patterns of molecular divergences in Tuatara mitogenomes.

Population genetic models predict that populations that are geographically close to each other are expected to be genetically more similar to each other compared to those that are widely separate. However the patterns of relationships between geographic distance and molecular divergences at neutral and constrained regions of the genome are unclear. We attempted to clarify this relationship by sequencing complete mitochondrial genomes of the relic species Tuatara (Sphenodon punctatus) from ten offshore islands of New Zealand. We observed a positive relationship that showed a proportional increase in the neutral diversity at synonymous sites (dS), with increasing geographical distance. In contrast we showed that diversity at evolutionarily constrained sites (dC) was elevated in the case of comparisons involving closely located populations. Conversely diversity was reduced in the case of comparisons between distantly located populations. These patterns were confirmed by a significant negative relationship between the ratio of dC/dS and geographic distance. The observed high dC/dS could be explained by the abundance of deleterious mutations in comparisons involving closely located populations, due to the recent population divergence times. Since distantly related populations were separated over long periods of time, deleterious mutations might have been removed by purifying selection.

Complete mitochondrial genomes of Tuatara endemic to different islands of New Zealand.

Tuatara are the sister taxon to the Squamata (including lizards and snakes) and are regarded as the most distinctive surviving reptilian genus. They are currently inhabits on offshore islands around New Zealand and have been recognized as a species in need of active conservation management. In this study, we report a total number of five nearly complete mitochondrial genomes, which were sequenced by Sanger and Next Generation DNA sequencing methods. Our phylogenomic analysis revealed distinct clustering of tuatara populations from the north and south islands of New Zealand.

The mysterious Spotted Green Pigeon and its relation to the Dodo and its kindred.

BACKGROUND: The closely related and extinct Dodo (Raphus cucullatus) and Rodrigues Solitaire (Pezophaps solitaria), both in the subfamily Raphinae, are members of a clade of morphologically very diverse pigeons. Genetic analyses have revealed that the Nicobar Pigeon (Caloenas nicobarica) is the closest living relative of these birds, thereby highlighting their ancestors' remarkable migration and morphological evolution. The Spotted Green Pigeon (Caloenas maculata) was described in 1783 and showed some similarities to the Nicobar Pigeon. Soon however the taxon fell into obscurity, as it was regarded as simply an abnormal form of the Nicobar Pigeon. The relationship between both taxa has occasionally been questioned, leading some ornithologists to suggest that the two may in fact be different taxa. Today only one of the original two specimens survives and nothing is known about the origin of the taxon. Due to its potential close relationship, the Spotted Green Pigeon may hold clues to the historical migration, isolation and morphological evolution of the Dodo and its kindred. RESULTS: We use ancient DNA methodologies to investigate the phylogeny and authenticity of the Spotted Green Pigeon. A novel extraction method with the ability to retain and purify heavily fragmented DNA is used to investigate two feathers from the sole surviving specimen. Maximum Likelihood phylogenetic analyses reveal that the Spotted Green Pigeon is a unique lineage and together with the Nicobar Pigeon, is basal to the Dodo and Rodrigues Solitaire. CONCLUSIONS: The distance observed for the Spotted Green Pigeon and Nicobar Pigeon is larger than that observed within other Pigeon species, indicating that the Spotted Green pigeon is a unique taxon, thereby also indicating it is a genuine addition to the list of extinct species. The phylogenetic placement of the Spotted Green Pigeon indicates that the ancestors of both Caloenas and therefore Raphinae displayed and shared the following traits: ability of flight, semi-terrestrial habits and an affinity towards islands. This set of traits supports the stepping stone hypothesis, which states that the Raphinae got to their respective localities by island hopping from India or Southeast Asia.

Reconstruction and in vivo analysis of the extinct tbx5 gene from ancient wingless moa (Aves: Dinornithiformes).

BACKGROUND: The forelimb-specific gene tbx5 is highly conserved and essential for the development of forelimbs in zebrafish, mice, and humans. Amongst birds, a single order, Dinornithiformes, comprising the extinct wingless moa of New Zealand, are unique in having no skeletal evidence of forelimb-like structures. RESULTS: To determine the sequence of tbx5 in moa, we used a range of PCR-based techniques on ancient DNA to retrieve all nine tbx5 exons and splice sites from the giant moa, Dinornis. Moa Tbx5 is identical to chicken Tbx5 in being able to activate the downstream promotors of fgf10 and ANF. In addition we show that missexpression of moa tbx5 in the hindlimb of chicken embryos results in the formation of forelimb features, suggesting that Tbx5 was fully functional in wingless moa. An alternatively spliced exon 1 for tbx5 that is expressed specifically in the forelimb region was shown to be almost identical between moa and ostrich, suggesting that, as well as being fully functional, tbx5 is likely to have been expressed normally in moa since divergence from their flighted ancestors, approximately 60 mya. CONCLUSIONS: The results suggests that, as in mice, moa tbx5 is necessary for the induction of forelimbs, but is not sufficient for their outgrowth. Moa Tbx5 may have played an important role in the development of moa's remnant forelimb girdle, and may be required for the formation of this structure. Our results further show that genetic changes affecting genes other than tbx5 must be responsible for the complete loss of forelimbs in moa.

Identification, classification, and growth of moa chicks (Aves: Dinornithiformes) from the genus Euryapteryx.

BACKGROUND: The analysis of growth in extinct organisms is difficult. The general lack of skeletal material from a range of developmental states precludes determination of growth characteristics. For New Zealand's extinct moa we have available to us a selection of rare femora at different developmental stages that have allowed a preliminary determination of the early growth of this giant flightless bird. We use a combination of femora morphometrics, ancient DNA, and isotope analysis to provide information on the identification, classification, and growth of extinct moa from the genus Euryapteryx. RESULTS: Using ancient DNA, we identify a number of moa chick bones for the species Euryapteryx curtus, Dinornis novaezealandiae, and Anomalopteryx didiformis, and the first chick bone for Pachyornis geranoides. Isotope analysis shows that ∂15N levels vary between the two known size classes of Euryapteryx, with the larger size class having reduced levels of ∂15N. A growth series for femora of the two size classes of Euryapteryx shows that early femora growth characteristics for both classes are almost identical. Morphometric, isotopic, and radiographic analysis of the smallest Euryapteryx bones suggests that one of these femora is from a freshly hatched moa at a very early stage of development. CONCLUSION: Using morphometric, isotopic, and ancient DNA analyses have allowed the determination of a number of characteristics of rare moa chick femora. For Euryapteryx the analyses suggest that the smaller sized class II Euryapteryx is identical in size and growth to the extant Darwin's rhea.

Complex species status for extinct moa (Aves: Dinornithiformes) from the genus Euryapteryx.

The exact species status of New Zealand's extinct moa remains unknown. In particular, moa belonging to the genus Euryapteryx have been difficult to classify. We use the DNA barcoding sequence on a range of Euryapteryx samples in an attempt to resolve the species status for this genus. We obtained mitochondrial control region and the barcoding region from Cytochrome Oxidase Subunit I (COI) from a number of new moa samples and use available sequences from previous moa phylogenies and eggshell data to try and clarify the species status of Euryapteryx. Using the COI barcoding region we show that species status in Euryapteryx is complex with no clear separation between various individuals. Eggshell, soil, and bone data suggests that a Euryapteryx subspecies likely exists on New Zealand's North Island and can be characterized by a single mitochondrial control region SNP. COI divergences between Euryapteryx individuals from the south of New Zealand's South Island and those from the Far North of the North Island exceed 1.6% and are likely to represent separate species. Individuals from other areas of New Zealand were unable to be clearly separated based on COI differences possibly as a result of repeated hybridisation events. Despite the accuracy of the COI barcoding region to determine species status in birds, including that for the other moa genera, for moa from the genus Euryapteryx, COI barcoding fails to provide a clear result, possibly as a consequence of repeated hybridisation events between these moa. A single control region SNP was identified however that segregates with the two general morphological variants determined for Euryapteryx; a smaller subspecies restricted to the North Island of New Zealand, and a larger subspecies, found on both New Zealand's North and South Island.

DNA fingerprinting in zoology: past, present, future.

In 1962, Thomas Kuhn famously argued that the progress of scientific knowledge results from periodic 'paradigm shifts' during a period of crisis in which new ideas dramatically change the status quo. Although this is generally true, Alec Jeffreys' identification of hypervariable repeat motifs in the human beta-globin gene, and the subsequent development of a technology known now as 'DNA fingerprinting', also resulted in a dramatic shift in the life sciences, particularly in ecology, evolutionary biology, and forensics. The variation Jeffreys recognized has been used to identify individuals from tissue samples of not just humans, but also of many animal species. In addition, the technology has been used to determine the sex of individuals, as well as paternity/maternity and close kinship. We review a broad range of such studies involving a wide diversity of animal species. For individual researchers, Jeffreys' invention resulted in many ecologists and evolutionary biologists being given the opportunity to develop skills in molecular biology to augment their whole organism focus. Few developments in science, even among the subsequent genome discoveries of the 21st century, have the same wide-reaching significance. Even the later development of PCR-based genotyping of individuals using microsatellite repeats sequences, and their use in determining multiple paternity, is conceptually rooted in Alec Jeffreys' pioneering work.

Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment.

BACKGROUND: Penguins are flightless aquatic birds widely distributed in the Southern Hemisphere. The distinctive morphological and physiological features of penguins allow them to live an aquatic life, and some of them have successfully adapted to the hostile environments in Antarctica. To study the phylogenetic and population history of penguins and the molecular basis of their adaptations to Antarctica, we sequenced the genomes of the two Antarctic dwelling penguin species, the Adélie penguin [Pygoscelis adeliae] and emperor penguin [Aptenodytes forsteri]. RESULTS: Phylogenetic dating suggests that early penguins arose ~60 million years ago, coinciding with a period of global warming. Analysis of effective population sizes reveals that the two penguin species experienced population expansions from ~1 million years ago to ~100 thousand years ago, but responded differently to the climatic cooling of the last glacial period. Comparative genomic analyses with other available avian genomes identified molecular changes in genes related to epidermal structure, phototransduction, lipid metabolism, and forelimb morphology. CONCLUSIONS: Our sequencing and initial analyses of the first two penguin genomes provide insights into the timing of penguin origin, fluctuations in effective population sizes of the two penguin species over the past 10 million years, and the potential associations between these biological patterns and global climate change. The molecular changes compared with other avian genomes reflect both shared and diverse adaptations of the two penguin species to the Antarctic environment.

Evidence for a recent origin of penguins.

Penguins are a remarkable group of birds, with the 18 extant species living in diverse climatic zones from the tropics to Antarctica. The timing of the origin of these extant penguins remains controversial. Previous studies based on DNA sequences and fossil records have suggested widely differing times for the origin of the group. This has given rise to widely differing biogeographic narratives about their evolution. To resolve this problem, we sequenced five introns from 11 species representing all genera of living penguins. Using these data and other available DNA sequences, together with the ages of multiple penguin fossils to calibrate the molecular clock, we estimated the age of the most recent common ancestor of extant penguins to be 20.4 Myr (17.0-23.8 Myr). This time is half of the previous estimates based on molecular sequence data. Our results suggest that most of the major groups of extant penguins diverged 11-16 Ma. This overlaps with the sharp decline in Antarctic temperatures that began approximately 12 Ma, suggesting a possible relationship between climate change and penguin evolution.