Ancient proteins resolve controversy over the identity of Genyornis eggshell.

The realization that ancient biomolecules are preserved in "fossil" samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia's indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller et al. [Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.

Subsistence practices, past biodiversity, and anthropogenic impacts revealed by New Zealand-wide ancient DNA survey.

New Zealand's geographic isolation, lack of native terrestrial mammals, and Gondwanan origins make it an ideal location to study evolutionary processes. However, since the archipelago was first settled by humans 750 y ago, its unique biodiversity has been under pressure, and today an estimated 49% of the terrestrial avifauna is extinct. Current efforts to conserve the remaining fauna rely on a better understanding of the composition of past ecosystems, as well as the causes and timing of past extinctions. The exact temporal and spatial dynamics of New Zealand's extinct fauna, however, can be difficult to interpret, as only a small proportion of animals are preserved as morphologically identifiable fossils. Here, we conduct a large-scale genetic survey of subfossil bone assemblages to elucidate the impact of humans on the environment in New Zealand. By genetically identifying more than 5,000 nondiagnostic bone fragments from archaeological and paleontological sites, we reconstruct a rich faunal record of 110 species of birds, fish, reptiles, amphibians, and marine mammals. We report evidence of five whale species rarely reported from New Zealand archaeological middens and characterize extinct lineages of leiopelmatid frog (Leiopelma sp.) and kakapo (Strigops habroptilus) haplotypes lost from the gene pool. Taken together, this molecular audit of New Zealand's subfossil record not only contributes to our understanding of past biodiversity and precontact Maori subsistence practices but also provides a more nuanced snapshot of anthropogenic impacts on native fauna after first human arrival.

Eggshell palaeogenomics: Palaeognath evolutionary history revealed through ancient nuclear and mitochondrial DNA from Madagascan elephant bird (Aepyornis sp.) eggshell.

Palaeognaths, the sister group of all other living birds (neognaths), were once considered to be vicariant relics from the breakup of the Gondwanan supercontinent. However, recent molecular studies instead argue for dispersal of volant ancestors across marine barriers. Resolving this debate hinges upon accurately reconstructing their evolutionary relationships and dating their divergences, which often relies on phylogenetic information from extinct relatives and nuclear genomes. Mitogenomes from the extinct elephant birds of Madagascar have helped inform the palaeognath phylogeny; however, nuclear information has remained unavailable. Here, we use ancient DNA (aDNA) extracted from fossil eggshell, together with target enrichment and next-generation sequencing techniques, to reconstruct an additional new mitogenome from Aepyornis sp. with 33.5X coverage. We also recover the first elephant bird nuclear aDNA, represented by 12,500bp of exonic information. While we confirm that elephant birds are sister taxa to the kiwi, our data suggests that, like neognaths, palaeognaths underwent an explosive radiation between 69 and 52Ma-well after the break-up of Gondwana, and more rapidly than previously estimated from mitochondrial data alone. These results further support the idea that ratites primarily diversified immediately following the Cretaceous-Palaeogene mass extinction and convergently evolved flightlessness. Our study reinforces the importance of including information from the nuclear genome of extinct taxa for recovering deep evolutionary relationships. Furthermore, with approximately 3% endogenous aDNA retrieved, avian eggshell can be a valuable substrate for recovering high quality aDNA. We suggest that elephant bird whole genome recovery is ultimately achievable, and will provide future insights into the evolution these birds.

Strong extrinsic reproductive isolation between parapatric populations of an Australian groundsel.

Speciation with gene flow, or the evolution of reproductive isolation between interbreeding populations, remains a controversial problem in evolution. This is because gene flow erodes the adaptive differences that selection creates between populations. Here, we use a combination of common garden experiments in the field and in the glasshouse to investigate what ecological and genetic mechanisms prevent gene flow and maintain morphological and genetic differentiation between coastal parapatric populations of the Australian groundsel Senecio lautus. We discovered that in each habitat extrinsic reproductive barriers prevented gene flow, whereas intrinsic barriers in F1 hybrids were weak. In the field, herbivores played a major role in preventing gene flow, but glasshouse experiments demonstrated that soil type also created variable selective pressures both locally and on a greater geographic scale. Our experimental results demonstrate that interfertile plant populations adapting to contrasting environments may diverge as a consequence of concurrent natural selection acting against migrants and hybrids through multiple mechanisms. These results provide novel insights into the consequences of local adaptation in the origin of strong barriers to gene flow in plants, and suggest that herbivory may play an important role in the early stages of plant speciation.

Molecular exploration of fossil eggshell uncovers hidden lineage of giant extinct bird.

The systematics of Madagascar's extinct elephant birds remains controversial due to large gaps in the fossil record and poor biomolecular preservation of skeletal specimens. Here, a molecular analysis of 1000-year-old fossil eggshells provides the first description of elephant bird phylogeography and offers insight into the ecology and evolution of these flightless giants. Mitochondrial genomes from across Madagascar reveal genetic variation that is correlated with eggshell morphology, stable isotope composition, and geographic distribution. The elephant bird crown is dated to ca. 30 Mya, when Madagascar is estimated to have become less arid as it moved northward. High levels of between-clade genetic variation support reclassifying Mullerornis into a separate family. Low levels of within-clade genetic variation suggest there were only two elephant bird genera existing in southern Madagascar during the Holocene. However, we find an eggshell collection from Madagascar's far north that represents a unique lineage of Aepyornis. Furthermore, divergence within Aepyornis coincides with the aridification of Madagascar during the early Pleistocene ca. 1.5 Ma, and is consistent with the fragmentation of populations in the highlands driving diversification and the evolution of extreme gigantism over shorts timescales. We advocate for a revision of their taxonomy that integrates palaeogenomic and palaeoecological perspectives.

Thicker eggshells are not predicted by host egg ejection behaviour in four species of Australian cuckoo.

Defences of hosts against brood parasitic cuckoos include detection and ejection of cuckoo eggs from the nest. Ejection behaviour often involves puncturing the cuckoo egg, which is predicted to drive the evolution of thicker eggshells in cuckoos that parasitise such hosts. Here we test this prediction in four Australian cuckoo species and their hosts, using Hall-effect magnetic-inference to directly estimate eggshell thickness in parasitised clutches. In Australia, hosts that build cup-shaped nests are generally adept at ejecting cuckoo eggs, whereas hosts that build dome-shaped nests mostly accept foreign eggs. We analysed two datasets: a small sample of hosts with known egg ejection rates and a broader sample of hosts where egg ejection behaviour was inferred based on nest type (dome or cup). Contrary to predictions, cuckoos that exploit dome-nesting hosts (acceptor hosts) had significantly thicker eggshells relative to their hosts than cuckoos that exploit cup-nesting hosts (ejector hosts). No difference in eggshell thicknesses was observed in the smaller sample of hosts with known egg ejection rates, probably due to lack of power. Overall cuckoo eggshell thickness did not deviate from the expected avian relationship between eggshell thickness and egg length estimated from 74 bird species. Our results do not support the hypothesis that thicker eggshells have evolved in response to host ejection behaviour in Australian cuckoos, but are consistent with the hypothesis that thicker eggshells have evolved to reduce the risk of breakage when eggs are dropped into dome nests.

Unlocking inaccessible historical genomes preserved in formalin.

Museum specimens represent an unparalleled record of historical genomic data. However, the widespread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1962 and 2006 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence. We recovered complete mitochondrial genomes and up to 3× nuclear genome coverage from formalin-preserved tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out-performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation. Here, we demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and nondestructive assessment criteria. Sequencing of formalin-preserved specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.

Genetic barcoding of museum eggshell improves data integrity of avian biological collections.

Natural history collections are often plagued by missing or inaccurate metadata for collection items, particularly for specimens that are difficult to verify or rare. Avian eggshell in particular can be challenging to identify due to extensive morphological ambiguity among taxa. Species identifications can be improved using DNA extracted from museum eggshell; however, the suitability of current methods for use on small museum eggshell specimens has not been rigorously tested, hindering uptake. In this study, we compare three sampling methodologies to genetically identify 45 data-poor eggshell specimens, including a putatively extinct bird's egg. Using an optimised drilling technique to retrieve eggshell powder, we demonstrate that sufficient DNA for molecular identification can be obtained from even the tiniest eggshells without significant alteration to the specimen's appearance or integrity. This method proved superior to swabbing the external surface or sampling the interior; however, we also show that these methods can be viable alternatives. We then applied our drilling method to confirm that a purported clutch of Paradise Parrot eggs collected 40 years after the species' accepted extinction date were falsely identified, laying to rest a 53-year-old ornithological controversy. Thus, even the smallest museum eggshells can offer new insights into old questions.

Museum Epigenomics: Charting the Future by Unlocking the Past.

Epigenomic state preserved in museum specimens could be leveraged to provide unique insights into gene regulation trends associated with accelerating environmental change during the Anthropocene. We address the challenges facing museum epigenomics and propose a collaborative framework for researchers and curators to explore this new field.

Under the karst: detecting hidden subterranean assemblages using eDNA metabarcoding in the caves of Christmas Island, Australia.

Subterranean ecosystems are understudied and challenging to conventionally survey given the inaccessibility of underground voids and networks. In this study, we conducted a eukaryotic environmental DNA (eDNA) metabarcoding survey across the karst landscape of Christmas Island, (Indian Ocean, Australia) to evaluate the utility of this non-invasive technique to detect subterranean aquatic 'stygofauna' assemblages. Three metabarcoding assays targeting the mitochondrial 16S rRNA and nuclear 18S genes were applied to 159 water and sediment samples collected from 23 caves and springs across the island. Taken together, our assays detected a wide diversity of chordates, cnidarians, porifera, arthropods, molluscs, annelids and bryozoans from 71 families across 60 orders. We report a high level of variation between cave and spring subterranean community compositions which are significantly influenced by varying levels of salinity. Additionally, we show that dissolved oxygen and longitudinal gradients significantly affect biotic assemblages within cave communities. Lastly, we combined eDNA-derived community composition and environmental (water quality) data to predict potential underground interconnectivity across Christmas Island. We identified three cave and spring groups that showed a high degree of biotic and abiotic similarity indicating likely local connectivity. This study demonstrates the applicability of eDNA metabarcoding to detect subterranean eukaryotic communities and explore underground interconnectivity.

Rapid range shifts and megafaunal extinctions associated with late Pleistocene climate change.

Large-scale changes in global climate at the end of the Pleistocene significantly impacted ecosystems across North America. However, the pace and scale of biotic turnover in response to both the Younger Dryas cold period and subsequent Holocene rapid warming have been challenging to assess because of the scarcity of well dated fossil and pollen records that covers this period. Here we present an ancient DNA record from Hall's Cave, Texas, that documents 100 vertebrate and 45 plant taxa from bulk fossils and sediment. We show that local plant and animal diversity dropped markedly during Younger Dryas cooling, but while plant diversity recovered in the early Holocene, animal diversity did not. Instead, five extant and nine extinct large bodied animals disappeared from the region at the end of the Pleistocene. Our findings suggest that climate change affected the local ecosystem in Texas over the Pleistocene-Holocene boundary, but climate change on its own may not explain the disappearance of the megafauna at the end of the Pleistocene.

Avian mitochondrial genomes retrieved from museum eggshell.

Avian eggshell is a bio-ceramic material with exceptional properties for preserving DNA within its crystalline structure, presenting an opportunity to retrieve genomic information from extinct or historical populations of birds. However, intracrystalline DNA has only been recovered from the large, thick eggshell of palaeognaths; members of their more-diverse sister group (neognaths) lay smaller, thinner eggs that may not exhibit the same propensity for DNA preservation. Here, we use three 40-60-year-old museum eggshell specimens of Australian neognath birds to determine the minimum mass of eggshell from which intracrystalline DNA can be retrieved, and to characterize the yield and quality of such DNA. In doing so, we describe the first protocol for successful extraction of intracrystalline DNA from neognath eggshells, with the view to unlocking the potential of vast museum egg collections for genetic research. We were able to retrieve DNA fragments over 200 bp in length from 10 mg of eggshell powder from all three specimens, and demonstrate that expanding the existing blow-hole can allow sufficient material to be collected for DNA extraction while minimizing damage to the appearance and structural integrity of the egg. Furthermore, we were able to reconstruct near-complete mitochondrial genomes at a coverage of 40-83X through shotgun sequencing of these extracts on three NextSeq lanes. Given the current extinction and extirpation rates of many avian species world-wide, genetic data from eggshell could provide a rapid and cost-effective approach to examining temporal changes in avian diversity, which is not only becoming crucial for conservation management, but also serve to deepen our understanding of genome-wide evolutionary processes.

Time to Spread Your Wings: A Review of the Avian Ancient DNA Field.

Ancient DNA (aDNA) has the ability to inform the evolutionary history of both extant and extinct taxa; however, the use of aDNA in the study of avian evolution is lacking in comparison to other vertebrates, despite birds being one of the most species-rich vertebrate classes. Here, we review the field of "avian ancient DNA" by summarising the past three decades of literature on this topic. Most studies over this time have used avian aDNA to reconstruct phylogenetic relationships and clarify taxonomy based on the sequencing of a few mitochondrial loci, but recent studies are moving toward using a comparative genomics approach to address developmental and functional questions. Applying aDNA analysis with more practical outcomes in mind (such as managing conservation) is another increasingly popular trend among studies that utilise avian aDNA, but the majority of these have yet to influence management policy. We find that while there have been advances in extracting aDNA from a variety of avian substrates including eggshell, feathers, and coprolites, there is a bias in the temporal focus; the majority of the ca. 150 studies reviewed here obtained aDNA from late Holocene (100-1000 yBP) material, with few studies investigating Pleistocene-aged material. In addition, we identify and discuss several other issues within the field that require future attention. With more than one quarter of Holocene bird extinctions occurring in the last several hundred years, it is more important than ever to understand the mechanisms driving the evolution and extinction of bird species through the use of aDNA.

DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea).

The analysis of apex predator diet has the ability to deliver valuable insights into ecosystem health, and the potential impacts a predator might have on commercially relevant species. The Australian sea lion (Neophoca cinerea) is an endemic apex predator and one of the world's most endangered pinnipeds. Given that prey availability is vital to the survival of top predators, this study set out to understand what dietary information DNA metabarcoding could yield from 36 sea lion scats collected across 1,500 km of its distribution in southwest Western Australia. A combination of PCR assays were designed to target a variety of potential sea lion prey, including mammals, fish, crustaceans, cephalopods, and birds. Over 1.2 million metabarcodes identified six classes from three phyla, together representing over 80 taxa. The results confirm that the Australian sea lion is a wide-ranging opportunistic predator that consumes an array of mainly demersal fauna. Further, the important commercial species Sepioteuthis australis (southern calamari squid) and Panulirus cygnus (western rock lobster) were detected, but were present in <25% of samples. Some of the taxa identified, such as fish, sharks and rays, clarify previous knowledge of sea lion prey, and some, such as eel taxa and two gastropod species, represent new dietary insights. Even with modest sample sizes, a spatial analysis of taxa and operational taxonomic units found within the scat shows significant differences in diet between many of the sample locations and identifies the primary taxa that are driving this variance. This study provides new insights into the diet of this endangered predator and confirms the efficacy of DNA metabarcoding of scat as a noninvasive tool to more broadly define regional biodiversity.

Genetic variants in the ETV5 gene in fertile and infertile men with nonobstructive azoospermia associated with Sertoli cell-only syndrome.

OBJECTIVE: To assess the association between genetic variants in the ETV5 gene with nonobstructive azoospermia (NOA) associated with Sertoli cell-only (SCO) syndrome. DESIGN: Genetic association study. SETTING: University. PATIENT(S): Australian men (65 SCO, 53 NOA, and 242 fertile men) and American men (86 SCO and 54 fertile men). INTERVENTION(S): Paraffin-embedded human testicular tissue was sectioned and processed for immunofluorescence. Direct DNA sequencing and polymerase chain reaction-based SNP detection were performed to define genetic variants in the ETV5 gene. MAIN OUTCOME MEASURE(S): The localization of ETV5 in the human testis and the presence of ETV5 genetic variants in fertile and infertile men. RESULT(S): ETV5 is localized to the cytoplasm and nucleus of Sertoli and germ cells in adult human testes. We identified six previously reported and six new genetic variants in the ETV5 gene. Of these, the allele frequency of the homozygous +48845 G>T (TT allele) variant was significantly higher in the SCO and NOA Australian men compared with fertile men. CONCLUSION(S): The homozygous +48845 G>T (TT allele) variant confers a higher risk for male infertility associated with NOA and SCO in Australian men.

The genetics and ecology of reinforcement: implications for the evolution of prezygotic isolation in sympatry and beyond.

Reinforcement, the evolution of prezygotic reproductive barriers by natural selection in response to maladaptive hybridization, is one of the most debated processes in speciation. Critics point to "fatal" conceptual flaws for sympatric evolution of prezygotic isolation, but recent theoretical and empirical work on genetics and ecology of reinforcement suggests that such criticisms can be overcome. New studies provide evidence for reinforcement in frogs, fish, insects, birds, and plants. While such evidence lays to rest the argument over reinforcement's existence, our understanding remains incomplete. We lack data on (1) the genetic basis of female preferences and the links between genetics of pre- and postzygotic isolation, (2) the ecological basis of reproductive isolation, (3) connections between prezygotic isolation between species and within-species sexual selection (potentially leading to a "cascade" of effects on reproductive isolation), (4) the role of habitat versus mate preference in reinforcement, and (5) additional detailed comparative studies. Here, we review data on these issues and highlight why they are important for understanding speciation.