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.

A Unique Mitochondrial Gene Block Inversion in Antarctic Trematomin Fishes: A Cautionary Tale.

Many Antarctic notothenioid fishes have major rearrangements in their mitochondrial (mt) genomes. Here, we report the complete mt genomes of 3 trematomin notothenioids: the bald notothen (Trematomus (Pagothenia) borchgrevinki), the spotted notothen (T. nicolai), and the emerald notothen (T. bernacchii). The 3 mt genomes were sequenced using next-generation Illumina technology, and the assemblies verified by Sanger sequencing. When compared with the canonical mt gene order of the Antarctic silverfish (Pleuragramma antarctica), we found a large gene inversion in the 3 trematomin mt genomes that included tRNAIle, ND1, tRNALeu2, 16S, tRNAVal, 12S, tRNAPhe, and the control region. The trematomin mt genomes contained 3 intergenic spacers, which are thought to be the remnants of previous gene and control region duplications. All control regions included the characteristic conserved regulatory sequence motifs. Although short-read next-generation DNA sequencing technology has allowed the rapid and cost-effective sequencing of a large number of complete mt genomes, it is essential in all cases to verify the assembly in order to prevent the publication and use of erroneous data.

The design and application of a 50 K SNP chip for a threatened Aotearoa New Zealand passerine, the hihi.

Next-generation sequencing has transformed the fields of ecological and evolutionary genetics by allowing for cost-effective identification of genome-wide variation. Single nucleotide polymorphism (SNP) arrays, or "SNP chips", enable very large numbers of individuals to be consistently genotyped at a selected set of these identified markers, and also offer the advantage of being able to analyse samples of variable DNA quality. We used reduced representation restriction-aided digest sequencing (RAD-seq) of 31 birds of the threatened hihi (Notiomystis cincta; stitchbird) and low-coverage whole genome sequencing (WGS) of 10 of these birds to develop an Affymetrix 50 K SNP chip. We overcame the limitations of having no hihi reference genome and a low quantity of sequence data by separate and pooled de novo assembly of each of the 10 WGS birds. Reads from all individuals were mapped back to these de novo assemblies to identify SNPs. A subset of RAD-seq and WGS SNPs were selected for inclusion on the chip, prioritising SNPs with the highest quality scores whose flanking sequence uniquely aligned to the zebra finch (Taeniopygia guttata) genome. Of the 58,466 SNPs manufactured on the chip, 72% passed filtering metrics and were polymorphic. By genotyping 1,536 hihi on the array, we found that SNPs detected in multiple assemblies were more likely to successfully genotype, representing a cost-effective approach to identify SNPs for genotyping. Here, we demonstrate the utility of the SNP chip by describing the high rates of linkage disequilibrium in the hihi genome, reflecting the history of population bottlenecks in the species.

Sex-specific foraging of an apex predator puts females at risk of human-wildlife conflict.

Urbanisation and anthropogenic alteration of ecosystems has led to conflict between humans and wildlife. Such conflict is often observed in apex predators. Although human-wildlife conflict has been extensively studied, male/female differences in behaviour are rarely considered. We investigated male/female differences in foraging behaviour of the predatory/scavenging brown skua Catharacta antarctica lonnbergi breeding on a New Zealand island nature reserve in proximity to farmland. These skuas are subject to culling, when perceived as a threat to livestock. As part of a long-term ecological study, we used high-resolution Global Positioning System (GPS) devices to characterise the space-use of foraging brown skuas. We also analysed stable isotopes of carbon (δ13 C) and nitrogen (δ15 N) from modern and archived blood samples to investigate possible changes in diet over the past ~30 years. Analysis of 100 GPS tracks collected from 2014 to 2016 demonstrated that males and females consistently visited different habitats. Males spent most of their time close to their breeding territory on the island nature reserve and females frequently visited a farmed island approximately two kilometres away. Consistent with this finding, we show that male and female skuas also differed markedly in their diets: males specialised on burrow-nesting white-faced storm petrels Pelagodroma marina (80%) with only a small proportion of sheep remains Ovis aries (<6%) contributing to their diet. In contrast, female diet comprised 27% white-faced storm petrels, other seabirds (18%) and a relatively large proportion of sheep remains (47%). Further, our data (186 blood samples from 122 individuals) show that this male/female difference in diet has persisted at least since 1987. Because females fed disproportionally on sheep remains, they may be more vulnerable to being culled by farmers. Importantly, our case study suggests that intersexual differences in diet and foraging patterns can have major implications for the reproduction and survival of apex predators that interact with farming. We strongly suggest that intersexual differences in behaviour should be considered when investigating human-wildlife conflicts.

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.

Genetic structure of the grey side-gilled sea slug (Pleurobranchaea maculata) in coastal waters of New Zealand.

Pleurobranchaea maculata is a rarely studied species of the Heterobranchia found throughout the south and western Pacific-and recently recorded in Argentina-whose population genetic structure is unknown. Interest in the species was sparked in New Zealand following a series of dog deaths caused by ingestions of slugs containing high levels of the neurotoxin tetrodotoxin. Here we describe the genetic structure and demographic history of P. maculata populations from five principle locations in New Zealand based on extensive analyses of 12 microsatellite loci and the COI and CytB regions of mitochondrial DNA (mtDNA). Microsatellite data showed significant differentiation between northern and southern populations with population structure being associated with previously described regional variations in tetrodotoxin concentrations. However, mtDNA sequence data did not support such structure, revealing a star-shaped haplotype network with estimates of expansion time suggesting a population expansion in the Pleistocene era. Inclusion of publicly available mtDNA sequence sea slugs from Argentina did not alter the star-shaped network. We interpret our data as indicative of a single founding population that fragmented following geographical changes that brought about the present day north-south divide in New Zealand waters. Lack of evidence of cryptic species supports data indicating that differences in toxicity of individuals among regions are a consequence of differences in diet.

Genetic Kinship Analyses Reveal That Gray's Beaked Whales Strand in Unrelated Groups.

Some marine mammals are so rarely seen that their life history and social structure remain a mystery. Around New Zealand, Gray's beaked whales (Mesoplodon grayi) are almost never seen alive, yet they are a commonly stranded species. Gray's are unique among the beaked whales in that they frequently strand in groups, providing an opportunity to investigate their social organization. We examined group composition and genetic kinship in 113 Gray's beaked whales with samples collected over a 20-year period. Fifty-six individuals stranded in 19 groups (2 or more individuals), and 57 whales stranded individually. Mitochondrial control region haplotypes and microsatellite genotypes (16 loci) were obtained for 103 whales. We estimated pairwise relatedness between all pairs of individuals and average relatedness within, and between, groups. We identified 6 mother-calf pairs and 2 half-siblings, including 2 whales in different strandings 17 years and 1500 km apart. Surprisingly, none of the adults stranding together were related suggesting that groups are not formed through the retention of kin. These data suggest that both sexes may disperse from their mothers, and groups consisting of unrelated subadults are common. We also found no instances of paternity within the groups. Our results provide the first insights into dispersal, social organization, and the mating system in this rarely sighted species. Why whales strand is still unknown but, in Gray's beaked whales, the dead can tell us much about the living.

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.

The effects of the general anaesthetic isoflurane on the honey bee (Apis mellifera) circadian clock.

General anaesthesia administered during the day has previously been shown to phase shift the honey bee clock. We describe a phase response curve for honey bees (n=105) to six hour isoflurane anaesthesia. The honey bee isoflurane PRC is "weak" with a delay portion (maximum shift of -1.88 hours, circadian time 0 - 3) but no advance zone. The isoflurane-induced shifts observed here are in direct opposition to those of light. Furthermore, concurrent administration of light and isoflurane abolishes the shifts that occur with isoflurane alone. Light may thus provide a means of reducing isoflurane-induced phase shifts.

High coverage of the complete mitochondrial genome of the rare Gray's beaked whale (Mesoplodon grayi) using Illumina next generation sequencing.

Using an Illumina platform, we shot-gun sequenced the complete mitochondrial genome of Gray's beaked whale (Mesoplodon grayi) to an average coverage of 152X. We performed a de novo assembly using SOAPdenovo2 and determined the total mitogenome length to be 16,347 bp. The nucleotide composition was asymmetric (33.3% A, 24.6% C, 12.6% G, 29.5% T) with an overall GC content of 37.2%. The gene organization was similar to that of other cetaceans with 13 protein-coding genes, 2 rRNAs (12S and 16S), 22 predicted tRNAs and 1 control region or D-loop. We found no evidence of heteroplasmy or nuclear copies of mitochondrial DNA in this individual. Beaked whales within the genus Mesoplodon are rarely seen at sea and their basic biology is poorly understood. These data will contribute to resolving the phylogeography and population ecology of this speciose group.

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.

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.

Way-finding in displaced clock-shifted bees proves bees use a cognitive map.

Mammals navigate by means of a metric cognitive map. Insects, most notably bees and ants, are also impressive navigators. The question whether they, too, have a metric cognitive map is important to cognitive science and neuroscience. Experimentally captured and displaced bees often depart from the release site in the compass direction they were bent on before their capture, even though this no longer heads them toward their goal. When they discover their error, however, the bees set off more or less directly toward their goal. This ability to orient toward a goal from an arbitrary point in the familiar environment is evidence that they have an integrated metric map of the experienced environment. We report a test of an alternative hypothesis, which is that all the bees have in memory is a collection of snapshots that enable them to recognize different landmarks and, associated with each such snapshot, a sun-compass-referenced home vector derived from dead reckoning done before and after previous visits to the landmark. We show that a large shift in the sun-compass rapidly induced by general anesthesia does not alter the accuracy or speed of the homeward-oriented flight made after the bees discover the error in their initial postrelease flight. This result rules out the sun-referenced home-vector hypothesis, further strengthening the now extensive evidence for a metric cognitive map in bees.

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.