Background

Analysis of ancient DNA has provided invaluable information on past ecologies, ancient populations, and extinct species. We used a short snippet of highly variable mitochondrial control region sequence from New Zealand’s moa to characterise a large number of bones previously intractable to DNA analysis as well as bone fragments from swamps to gain information about the haplotype diversity and phylogeography that existed in five moa species.

Methodology/Principal Findings

By targeting such ‘snippets’, we show that moa populations differed substantially in geographic structure that is likely to be related to population mobility and history. We show that populations of Pachyornis geranoides, Dinornis novaezealandiae, and Dinornis robustus were highly structured and some appear to have occupied the same geographic location for hundreds of thousands of years. In contrast, populations of the moa Anomalopteryx didiformis and Euryapteryx curtus were widespread, with specific populations of the latter occupying both the North and South Islands of New Zealand. We further show that for a specific area, in this case a North Island swamp, complete haplotype diversity and even sex can be recovered from collections of small, often discarded, bone fragments.

Conclusions/Significance

Short highly variable mitochondrial ‘snippets’ allow successful typing of environmentally damaged and fragmented skeletal material, and can provide useful information about ancient population diversity and structure without the need to sample valuable, whole bones often held by museums.

In contrast to molecular rates for neutral mitochondrial sequences, rates for constrained sites (including nonsynonymous sites, D-loop, and RNA) in the mitochondrial genome are known to vary with the time frame used for their estimation. Here, we examined this issue for the nuclear genomes using single-nucleotide polymorphisms (SNPs) from six complete human genomes of individuals belonging to different populations. We observed a strong time-dependent distribution of nonsynonymous SNPs (nSNPs) in highly constrained genes. Typically, the proportion of young nSNPs specific to a single population was found to be up to three times higher than that of the ancient nSNPs shared between diverse human populations. In contrast, this trend disappeared, and a uniform distribution of young and old nSNPs was observed in genes under relaxed selective constraints. This suggests that because mutations in constrained genes are highly deleterious, they are removed over time, resulting in a relative overabundance of young nSNPs. In contrast, mutations in genes under relaxed constraints are nearly neutral, which leads to similar proportions of young and old SNPs. These results could be useful to researchers aiming to select appropriate genes or genomic regions for estimating evolutionary rates and species or population divergence times.

Some previous studies have suggested that rates of evolution inferred using molecular sequences vary substantially depending on the time frame over which they are measured, whereas a number of other studies have argued against this proposition. We examined this issue by separating positions of primate mitochondrial genomes that are under different levels of selection constraints. Our results revealed an order of magnitude variation in the evolutionary rates at constrained sites (including nonsynonymous sites, D-loop, and RNA) and virtually an identical rate of evolution at synonymous sites, independent of the timescales over which they were estimated. Although the evolutionary rate at nonsynonymous sites obtained using the European (H1 haplogroup) mitogenomes is 9–15 times higher than that estimated using the human–chimpanzee pair, in contrast, the rates at synonymous sites are similar between these comparisons. We also show that the ratio of divergence at nonsynonymous to synonymous sites estimated using intra- and interspecific comparisons vary up to nine times, which corroborates our results independent of calibration times.

Background

The King Island Emu (Dromaius ater) of Australia is one of several extinct emu taxa whose taxonomic relationship to the modern Emu (D. novaehollandiae) is unclear. King Island Emu were mainly distinguished by their much smaller size and a reported darker colour compared to modern Emu.

Methodology and Results

We investigated the evolutionary relationships between the King Island and modern Emu by the recovery of both nuclear and mitochondrial DNA sequences from sub-fossil remains. The complete mitochondrial control (1,094 bp) and cytochrome c oxidase subunit I (COI) region (1,544 bp), as well as a region of the melanocortin 1 receptor gene (57 bp) were sequenced using a multiplex PCR approach. The results show that haplotypes for King Island Emu fall within the diversity of modern Emu.

Conclusions

These data show the close relationship of these emu when compared to other congeneric bird species and indicate that the King Island and modern Emu share a recent common ancestor. King Island emu possibly underwent insular dwarfism as a result of phenotypic plasticity. The close relationship between the King Island and the modern Emu suggests it is most appropriate that the former should be considered a subspecies of the latter. Although both taxa show a close genetic relationship they differ drastically in size. This study also suggests that rates of morphological and neutral molecular evolution are decoupled.

Background

Kiwi is a highly distinctive, flightless and endangered ratite bird endemic to New Zealand. To understand the patterns of molecular evolution of the nuclear protein-coding genes in brown kiwi (Apteryx australis mantelli) and to determine the timescale of avian history we sequenced a transcriptome obtained from a kiwi embryo using next generation sequencing methods. We then assembled the conserved protein-coding regions using the chicken proteome as a scaffold.

Results

Using 1,543 conserved protein coding genes we estimated the neutral evolutionary divergence between the kiwi and chicken to be ~45%, which is approximately equal to the divergence computed for the human-mouse pair using the same set of genes. A large fraction of genes was found to be under high selective constraint, as most of the expressed genes appeared to be involved in developmental gene regulation. Our study suggests a significant relationship between gene expression levels and protein evolution. Using sequences from over 700 nuclear genes we estimated the divergence between the two basal avian groups, Palaeognathae and Neognathae to be 132 million years, which is consistent with previous studies using mitochondrial genes.

Conclusions

The results of this investigation revealed patterns of mutation and purifying selection in conserved protein coding regions in birds. Furthermore this study suggests a relatively cost-effective way of obtaining a glimpse into the fundamental molecular evolutionary attributes of a genome, particularly when no closely related genomic sequence is available.

The extinct Huia (Heteralocha acutirostris) of New Zealand represents the most extreme example of beak dimorphism known in birds. We used a combination of nuclear genotyping methods, molecular sexing, and morphometric analyses of museum specimens collected in the late 19th and early 20th centuries to quantify the sexual dimorphism and population structure of this extraordinary species. We report that the classical description of Huia as having distinctive sex-linked morphologies is not universally correct. Four Huia, sexed as females had short beaks and, on this basis, were indistinguishable from males. Hence, we suggest it is likely that Huia males and females were indistinguishable as juveniles and that the well-known beak dimorphism is the result of differential beak growth rates in males and females. Furthermore, we tested the prediction that the social organisation and limited powers of flight of Huia resulted in high levels of population genetic structure. Using a suite of microsatellite DNA loci, we report high levels of genetic diversity in Huia, and we detected no significant population genetic structure. In addition, using mitochondrial hypervariable region sequences, and likely mutation rates and generation times, we estimated that the census population size of Huia was moderately high. We conclude that the social organization and limited powers of flight did not result in a highly structured population.

Background

The New Zealand quail, Coturnix novaezealandiae, was widespread throughout New Zealand until its rapid extinction in the 1870's. To date, confusion continues to exist concerning the identity of C. novaezealandiae and its phylogenetic relationship to Coturnix species in neighbouring Australia, two of which, C. ypsilophora and C. pectoralis, were introduced into New Zealand as game birds. The Australian brown quail, C. ypsilophora, was the only species thought to establish with current populations distributed mainly in the northern part of the North Island of New Zealand. Owing to the similarities between C. ypsilophora, C. pectoralis, and C. novaezealandiae, uncertainty has arisen over whether the New Zealand quail is indeed extinct, with suggestions that remnant populations of C. novaezealandiae may have survived on offshore islands.

Methodology/Principal Findings

Using fresh and historical samples of Coturnix sp. from New Zealand and Australia, DNA analysis of selected mitochondrial regions was carried out to determine phylogenetic relationships and species status. Results show that Coturnix sp. specimens from the New Zealand mainland and offshore island Tiritiri Matangi are not the New Zealand quail but are genetically identical to C. ypsilophora from Australia and can be classified as the same species. Furthermore, cytochrome b and COI barcoding analysis of the New Zealand quail and Australia's C. pectoralis, often confused in museum collections, show that they are indeed separate species that diverged approximately 5 million years ago (mya). Gross morphological analysis of these birds suggests a parallel loss of sustained flight with very little change in other phenotypic characters such as plumage or skeletal structure.

Conclusion/Significance

Ancient DNA has proved invaluable for the detailed analysis and identification of extinct and morphologically cryptic taxa such as that of quail and can provide insights into the timing of evolutionary changes that influence morphology.

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent.

Author Summary

Molecular evolutionary theory suggests that for neutral DNA sequences, rates of mutation and evolution should be equal. However, there has been considerable variation in empirical estimates of rates of molecular change in vertebrate animals, even for the same regions of the mitochondrial genome. A difficulty is that evolutionary rates estimated from ancient DNA and short-term mutation rates are not available for the same species. We present data on the rate of mutation of the mitochondrial hypervariable region in Adélie penguins from the Antarctic. All recorded mutations were heteroplasmic in mothers, and almost invariably, both genetic variants were passed to their offspring. We compared this rate of mutation to the rate of evolution estimated using serially preserved ancient remains. We show that these two estimates were not statistically different from each other.

The little spotted kiwi (Apteryx owenii) is a flightless ratite formerly found throughout New Zealand but now greatly reduced in distribution. Previous phylogeographic studies of the related brown kiwi (A. mantelli, A. rowi and A. australis), with which little spotted kiwi was once sympatric, revealed extremely high levels of genetic structuring, with mitochondrial DNA haplotypes often restricted to populations. We surveyed genetic variation throughout the present and pre-human range of little spotted kiwi by obtaining mitochondrial DNA sequences from contemporary and ancient samples. Little spotted kiwi and great spotted kiwi (A. haastii) formed a monophyletic clade sister to brown kiwi. Ancient samples of little spotted kiwi from the northern North Island, where it is now extinct, formed a lineage that was distinct from remaining little spotted kiwi and great spotted kiwi lineages, potentially indicating unrecognized taxonomic diversity. Overall, little spotted kiwi exhibited much lower levels of genetic diversity and structuring than brown kiwi, particularly through the South Island. Our results also indicate that little spotted kiwi (or at least hybrids involving this species) survived on the South Island mainland until more recently than previously thought.

Pulse oximetry has been shown to be accurate under steady state conditions. In this study, the accuracy of four pulse oximeters are evaluated and compared during outpatient general anesthesia for third molar extractions. The oximeters evaluated are the Nellcor N-100, the Ohmeda 3700, the Novametrix model 500, and the Bird 4400 portable pulse oximeter.

Ultralight general anesthesia for oral surgery presents a unique challenge for respiratory monitoring in that patients are often not intubated and commonly experience periods of hyper- and hypoventilation. Airway obstruction, apnea, and laryngospasm may occur easily and patients often vocalize and move during surgery. Because hypoxemia is the primary cause of morbidity and mortality during anesthesia, an accurate, continuous, and noninvasive monitor of oxygenation is critical to risk management.

Twenty ASA class I and II patients underwent outpatient general anesthesia for third molar removal using nitrous oxide-oxygen, midazolam, fentanyl, and methohexital. Arterial blood samples were obtained at five-minute intervals during anesthesia, as well as any time a desaturation of >5% occurred, for measurement of arterial SaO2 with an IL282 CO-Oximeter. These values were compared with simultaneously recorded saturations observed for each pulse oximeter. A total of 122 arterial samples were obtained over a range of PaO2 from 52-323 mm Hg and observed saturations of 70-100%.

The Bird 4400 portable pulse oximeter proved to be the most accurate and reliably predicted arterial saturation under these conditions (y = 1.03x - 2.8, r = 0.85). The Novametrix model 500 pulse oximeter also demonstrated a high degree of accuracy by linear regression analysis, but displayed the lowest correlation coefficient (spread of data points) overall (y = 0.97x + 2.8, r = 0.80.) The Nellcor N-100 pulse oximeter also proved to be highly accurate. (y = 1.05x - 4.1, r = 0.84.) In contrast, regression analysis of the observed saturations obtained with the Ohmeda 3700 pulse oximeter revealed that this unit significantly underestimated arterial saturation (y = 1.20x - 19.6, r = 0.83.)

This study demonstrates that despite the rigorous conditions imposed by outpatient general anesthesia for oral surgery, three of the pulse oximeters tested were linearly accurate in predicting arterial oxyhemoglobin saturation over the range of 70-100%. The Ohmeda 3700 was found to significantly underestimate arterial saturation.

Images