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Abstract Background Timely and informed public health responses to infectious diseases such as COVID-19 necessitate reliable information about infection dynamics. The case ascertainment rate (CAR), the proportion of infections that are reported as cases, is typically much less than one and varies with testing practices and behaviours, making reported cases unreliable as the sole source of data. The concentration of viral RNA in wastewater samples provides an alternate measure of infection prevalence that is not affected by clinical testing, healthcare-seeking behaviour or access to care. Methods We construct a state-space model with observed data of levels of SARS-CoV-2 in wastewater and reported case incidence and estimate the hidden states of the effective reproduction number, R, and CAR using sequential Monte Carlo methods. Results We analyse data from 1 January 2022 to 31 March 2023 from Aotearoa New Zealand. Our model estimates that R peaks at 2.76 (95% CrI 2.20, 3.83) around 18 February 2022 and the CAR peaks around 12 March 2022. We calculate that New Zealand’s second Omicron wave in July 2022 is similar in size to the first, despite fewer reported cases. We estimate that the CAR in the BA.5 Omicron wave in July 2022 is approximately 50% lower than in the BA.1/BA.2 Omicron wave in March 2022. Conclusions Estimating R, CAR, and cumulative number of infections provides useful information for planning public health responses and understanding the state of immunity in the population. This model is a useful disease surveillance tool, improving situational awareness of infectious disease dynamics in real-time.

Wildlife conflicts require robust quantitative data on incidence and impacts, particularly among species of conservation and cultural concern. We apply a multi-assay framework to quantify predation in a southeastern Australian scenario where complex management implications and calls for predator culling have grown despite a paucity of data on seabird predation by recovering populations of long-nosed fur seals (Arctocephalus forsteri). We apply two ecological surveillance techniques to analyze this predator’s diet – traditional morphometric (prey hard-part) and environmental DNA metabarcoding (genetic) analyses using an avian specific primer for the 12S ribosomal RNA (rRNA) gene – to provide managers with estimated predation incidence, number of seabird species impacted and inter-prey species relative importance to the predator. DNA metabarcoding identified additional seabird taxa and provided relative quantitative information where multiple prey species occur within a sample; while parallel use of both genetic and hard-part analyses revealed a greater diversity of taxa than either method alone. Using data from both assays, the estimated frequency of occurrence of predation on seabirds by long-nosed fur seals ranged from 9.1–29.3% of samples and included up to 6 detected prey species. The most common seabird prey was the culturally valued little penguin (Eudyptula minor) that occurred in 6.1–25.3% of samples, higher than previously reported from traditional morphological assays alone. We then explored DNA haplotype diversity for little penguin genetic data, as a species of conservation concern, to provide a preliminary estimate of the number of individuals consumed. Polymorphism analysis of consumed little penguin DNA identified five distinct mitochondrial haplotypes – representing a minimum of 16 individual penguins consumed across 10 fur seal scat samples (equivalent to 10.1% of samples). We recommend rapid uptake and development of cost-effective genetic techniques and broader spatiotemporal sampling of fur seal diets to further quantify predation and hotspots of concern for wildlife conflict management.

Invertebrate-derived DNA (iDNA) metabarcoding from carrion flies is a powerful, non-invasive tool that has value for assessing vertebrate diversity. However, unknowns exist around the factors that influence vertebrate detections, such as spatial limits to iDNA signals or if detections are influenced by taxonomic class or estimated biomass of the vertebrates of interest. Using a bulk-collection method, we captured flies from within a zoo and along transects extending 4 km away from this location. From 920 flies, we detected 28 vertebrate species. Of the 28 detected species, we identified 9 species kept at the zoo, 8 mammals and 1 bird, but no reptiles. iDNA detections were highly geographically localized, and only a few zoo animals were detected outside the zoo setting. However, due to the low number of detections in our dataset, we found no influence of the taxonomic group or the estimated biomass of animals on their detectability. Our data suggest that iDNA detections from bulk-collected carrion flies, at least in urban settings in Australia, are predominantly determined by geographic proximity to the sampling location. This study presents an important step in understanding how iDNA techniques can be used in biodiversity monitoring.

Abstract Analysis of environmental DNA (eDNA) has gained widespread usage for taxonomically based biodiversity assessment. While interest in applying noninvasive eDNA monitoring for population genetic assessments has grown, its usage in this sphere remains limited. One barrier to uptake is that the effectiveness of eDNA detection below the species level remains to be determined for multiple species and environments. Here, we test the utility of this emergent technology to obtain within‐species haplotypic variation of New Zealand (NZ) blackfoot pāua (Haliotis iris). We compare mitochondrial haplotype diversity recovered from marine eDNA samples against traditional tissue samples of blackfoot pāua collected at the same NZ coastal site. Targeting the ATP8‐ATP6 region, we recovered four mitochondrial haplotypes from eDNA versus six haplotypes from tissue samples. Three common haplotypes were recovered with both eDNA and tissue samples, while only one out of three rare haplotypes – represented in tissue samples by one individual each – was recovered with our eDNA methods. We demonstrate that eDNA monitoring is an effective tool for recovering common genetic diversity from pāua, although rare (

Abstract Background Genomic technologies have become routine in the surveillance and monitoring of the coronavirus disease 2019 (COVID-19) pandemic, as evidenced by the millions of SARS-CoV-2 sequences uploaded to international databases. Yet the ways in which these technologies have been applied to manage the pandemic are varied. Main text Aotearoa New Zealand was one of a small number of countries to adopt an elimination strategy for COVID-19, establishing a managed isolation and quarantine system for all international arrivals. To aid our response, we rapidly set up and scaled our use of genomic technologies to help identify community cases of COVID-19, to understand how they had arisen, and to determine the appropriate action to maintain elimination. Once New Zealand pivoted from elimination to suppression in late 2021, our genomic response changed to focusing on identifying new variants arriving at the border, tracking their incidence around the country, and examining any links between specific variants and increased disease severity. Wastewater detection, quantitation and variant detection were also phased into the response. Here, we explore New Zealand’s genomic journey through the pandemic and provide a high-level overview of the lessons learned and potential future capabilities to better prepare for future pandemics. Conclusions Our commentary is aimed at health professionals and decision-makers who might not be familiar with genetic technologies, how they can be used, and why this is an area with great potential to assist in disease detection and tracking now and in the future.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

Abstract Hybridisation and introgression of eukaryotic genomes can generate new species or subsume existing ones, with direct and indirect consequences for biodiversity. An understudied component of these evolutionary forces is their potentially rapid effect on host gut microbiomes, and whether these pliable microcosms may serve as early biological indicators of speciation. We address this hypothesis in a field study of angelfishes (genus Centropyge), which have one of the highest prevalence of hybridisation within coral reef fish. In our study region of the Eastern Indian Ocean, the parent fish species and their hybrids cohabit and display no differences in their diet, behaviour, and reproduction, often interbreeding in mixed harems. Despite this ecological overlap, we show that microbiomes of the parent species are significantly different from each other in form and function based on total community composition, supporting the division of parents into distinct species, despite the confounding effects of introgression acting to homogenize parent species identity at other molecular markers. The microbiome of hybrid individuals, on the other hand, are not significantly different to each of the parents, instead harbouring an intermediate community composition. These findings suggest that shifts in gut microbiomes may be an early indicator of speciation in hybridising species.

In tropical marine ecosystems, the coral-based diet of benthic-feeding reef fishes provides a window into the composition and health of coral reefs. In this study, for the first time, we compare multi-assay metabarcoding sequences of environmental DNA (eDNA) isolated from seawater and partially digested gut items from an obligate corallivore butterflyfish (Chaetodon lunulatus) resident to coral reef sites in the South China Sea. We specifically tested the proportional and statistical overlap of the different approaches (seawater vs gut content metabarcoding) in characterizing eukaryotic community composition on coral reefs. Based on 18S and ITS2 sequence data, which differed in their taxonomic sensitivity, we found that gut content detections were only partially representative of the eukaryotic communities detected in the seawater based on low levels of taxonomic overlap (3 to 21%) and significant differences between the sampling approaches. Overall, our results indicate that dietary metabarcoding of specialized feeders can be complimentary to, but is no replacement for, more comprehensive environmental DNA assays of reef environments that might include the processing of different substrates (seawater, sediment, plankton) or traditional observational surveys. These molecular assays, in tandem, might be best suited to highly productive but cryptic oceanic environments (kelp forests, seagrass meadows) that contain an abundance of organisms that are often small, epiphytic, symbiotic, or cryptic.

The evolution and systematics of Madagascar’s extinct elephant birds remains unclear. Here, the authors recover genetic, stable isotope, morphological, and geographic data from fossil eggshell to describe variation among clades, identifying cryptic diversity and potential drivers of speciation.

In March 2022, Aotearoa New Zealand re-opened its border allowing quarantine-free travel for many travellers. Here, the authors describe circulating Omicron sub-variants before and after the reopening of the border and show that the rate of viral introductions grew roughly linearly with the increase in daily international travel.

Abstract Environmental DNA (eDNA) is increasingly being used to assess community composition in marine ecosystems. Applying eDNA approaches across broad spatial scales now provide the potential to inform biogeographic analyses. However, to date, few studies have employed this technique to assess broad biogeographic patterns across multiple taxonomic groups. Here, we compare eDNA‐derived communities of bony fishes and invertebrates, including corals and sponges, from 15 locations spanning the entire length of the Omani coast. This survey includes a variety of habitats, including coral and rocky reefs, and covers three distinct marine ecoregions. Our data support a known biogeographic break in fish communities between the north and the south of Oman; however, the eDNA data highlight that this faunal break is mostly reflected in schooling baitfish species (e.g., sardines and anchovies), whereas reef‐associated fish communities appear more homogeneous along this coastline. Furthermore, our data provide indications that these biogeographic breaks also affect invertebrate communities, which includes corals, sponges, and broader eukaryotic groups. The observed community shifts were correlated with local environmental and anthropogenic differences characteristic of this coastline, particularly for the eDNA‐derived bony fish communities. Overall, this study provides compelling support that eDNA sequencing and associated analyses may serve as powerful tools to detect community differences across biogeographic breaks and ecoregions, particularly in places where there is significant variation in oceanographic conditions or anthropogenic impacts.

Abstract The need for efficient and more accurate ways of monitoring threatened ecosystems is becoming increasingly urgent as climate change intensifies. Coral reefs are an example of an ecosystem in crisis, with widespread declines in coral cover and diversity documented over recent decades. Novel molecular approaches such as biomonitoring using environmental DNA (eDNA) from seawater samples show great potential to complement future coral reef monitoring programs, especially when used in combination with conventional methods. However, eDNA metabarcoding studies often rely on public databases (e.g., GenBank) for assigning taxonomy, which generally limits the number of sequences that can be taxonomically identified. The extent to which building reference tissue sequences improves taxonomic resolution has yet to be fully examined. Here, we combined traditional coral reef monitoring data with eDNA assessments derived from seawater collected at the highly diverse Rowley Shoals in Western Australia. Using two ITS2 assays developed to target basal metazoan DNA and a reference database spiked with 70 local coral specimens, we identified 37 genera and 40 species from 56 1 L seawater samples. We identified considerable overlap of taxa with visual survey data and showed that assignment of amplicon sequence variants was significantly improved when “spiking” the taxonomic classifier with curated sequences of locally collected species. Our findings showcase the potential of eDNA metabarcoding for monitoring the biodiversity of reef corals and highlight the importance of custom reference sequence databases for improving taxonomic resolution in metabarcoding studies.

Laura Gajdzik et al. used population genomics, dietary DNA metabarcoding, and climate models to understand the influence of the 2010/2011 marine heatwave on the migration of rabbitfish from tropical environments into temperate environments in Western Australia. Their results reveal (i) little genetic differentiation and high migration rates between tropical residents and temperate vagrants, (ii) an overall diverse diet (including phytoplankton, local and widespread algae, and kelp), and (iii) projected suitable climate conditions for vagrants to establish in temperate marine waters.

Over the last decade, the microbiome has received increasing attention as a key factor in macroorganism fitness. Sustainable pest management requires an understanding of the complex microbial endophyte communities existing symbiotically within plants and the way synthetic pesticides interact with them. Fungal endophytes are known to benefit plant growth and fitness and may deter pests and diseases. Recent advances in high-throughput sequencing (HTS) have enabled integrative microbiome studies, especially in agricultural contexts. Here, we profile the fungal endophyte community in the phyllosphere of two barley (Hordeum vulgare) cultivars exposed to two systemic foliar fungicides using metabarcoding, an HTS tool that constructs community profiles from environmental DNA. We studied the fungal nuclear ribosomal large subunit D2 and internal transcribed spacer 2 (ITS2) DNA markers through a bioinformatics pipeline introduced here. We found 88 and 128 unique amplicon sequence variants (ASVs) using the D2 and ITS2 metabarcoding assays, respectively. With principal coordinate analysis and permutational analysis of variance, ASV diversity did not change in response to barley cultivar or fungicide treatment; however, the community structure of unsprayed plants did change between two collection times 8 days apart. The workflow described here can be applied to other microbiome studies in agriculture and we hope it encourages further research into crop microbiomes to improve agroecosystem management.

Abstract Environmental DNA (eDNA) metabarcoding, where specific DNA markers (barcodes) are purified from the environment, sequenced, and used to identify organisms, is revolutionizing biodiversity science. Researchers, industry, and governments are increasingly incorporating eDNA surveys into their toolkits for biomonitoring because of their high accuracy, taxonomically holistic lens, and ease of deployment. Yet, the full value of this rich and revolutionary technique is not being realized because eDNA data are rarely maintained in consistent formats or on open platforms that facilitate easy re‐use or integration with other data layers. Here, we discuss opportunities to improve the accessibility and “FAIRness” of eDNA records with the aim to maximize the value of this remarkable source of information on environmental state and change.

Abstract The Lund collection is one of the oldest subfossil collections in the world. The vast assemblage of subfossils was collected in the 1830s and 1840s by Peter Wilhelm Lund in Lagoa Santa, Brazil, and was shipped to Copenhagen in 1848, where it was stored in various locations around the city with little attention for the future preservation of the collection. So far, successful genetic research on the material collected by Lund has been limited to two samples of human petrous bone. However, less is known about the preservation conditions of the vast amounts of small and fragmentary bones stored in the collection. To address this, we studied ancient DNA from bulk bone samples of approximately 100 bone fragments from the P.W. Lund collection from boxes with varying degrees of physical preservation conditions. Using bulk bone metabarcoding, we found a high species diversity in all samples. In total, we identified 17 species, representing 11 mammals, two birds, one fish, and three frogs. Of these, two species are new to the collection. Collectively, these results exhibit the potential of future genetic studies on the famous P.W. Lund collection and suggest that the effects of poor storage conditions are probably negligible compared with the long‐term in situ degradation that specimens undergo before excavation.

Abstract 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.

Abstract Environmental DNA (eDNA) metabarcoding methods have demonstrated their potential as noninvasive techniques for the monitoring and conservation of marine fishes, including rare and endangered taxa. However, the majority of these investigations have focused on large‐bodied taxa such as sharks and sturgeons. In contrast, eDNA studies on small‐bodied cryptic taxa are much less common. As a case in point, seahorses (members of the Syngnathidae family) have never been detected by eDNA, despite the fact that globally there are 14 species classified as “Threatened” by the IUCN. Here, we critically evaluate the ability of two existing broad‐spectrum fish metabarcoding assays (MiFish and 16S Fish) and explore the efficacy of two newly designed fish metabarcoding assays (16S_FishSyn_Short and 16S_FishSyn_Long) to detect Syngnathidae amidst a wide spectrum of fish species. Furthermore, a custom Western Australian 16S rRNA fish database was created to increase the likelihood of correct taxonomic assignments. With the newly designed assays, we detected four Syngnathidae species in a targeted eDNA survey of the Perth metropolitan area (Western Australia). These detections include the seahorse species Hippocampus subelongatus and Hippocampus breviceps, which represents the first time seahorse species have been detected using eDNA. The existing MiFish and 16S Fish assays did not detect any Syngnathidae. This evaluation of all four fish metabarcoding assays reinforces the view that every PCR assay has “blind spots”. In the context of complex environmental samples, no assay is universal and false negatives will occur due to a combination of PCR efficacy, primer binding, assay sensitivity, degeneracies in the primers, template competition, and amplicon length. Taken together, these data indicate that eDNA methodologies, with ongoing optimizations, will become an integral part of monitoring small‐bodied cryptic taxa such as seahorses, gobies, and blennies and can assist in mapping species’ distributions and prioritizing conservation areas.

Abstract Fish biodiversity can be measured by capturing and then sequencing free DNA present in water. Such environmental DNA (eDNA) methods offer an effective, noninvasive tool for species diversity measurement, although standardized protocols are not yet developed. We investigate how metrics of fish biodiversity revealed through eDNA analysis of water are influenced by sampling volume. Water samples were collected from the intertidal reef of Browse Island, a tropical, remote island in the Timor Sea. Aliquots from a single 20,700‐ml sample and multiple 2,000‐ml samples were filtered in various volumes (25–2,000 ml) across two membrane sizes (0.20 and 0.45 µm). A fish metabarcoding assay was used to characterize the fish diversity within aliquots. All samples, except one, yielded fish DNA sequences. Two hundred and nine operational taxonomic units (cf. species) representing 48 fish families were identified from the complete collection of DNA contained in all samples, comparable to the 200 fish species detected using conventional surveys at this location. Notable additions from eDNA methods were cryptic and nocturnal fish species. Nevertheless, large differences in taxonomic composition (

The impact of late Pleistocene climate change on ecosystems has been hard to assess. Here, the authors sequence ancient DNA from Hall’s Cave, Texas and find that both plant and vertebrate diversity decreased with cooling, and though plant diversity recovered with rewarming, megafauna went extinct.

Abstract Background The utility of environmental DNA (eDNA) metabarcoding surveys to accurately detect species depends on the degree of DNA dispersal. Multiple marine studies have observed only minimal eDNA transport by horizontal water movement across small spatial scales, leading to the conclusion that spatially specific eDNA signals accurately resemble in‐field species assemblages along a horizontal axis. Marine communities, however, are also structured vertically according to depth. In marine environments displaying permanent water stratification, vertical zonation patterns may be more apparent and present on smaller spatial scales (i.e., meters) than horizontal community structuring. The scale at which eDNA signals differ along a vertical transect and the accuracy of eDNA metabarcoding in revealing naturally stratified communities have yet to be assessed. Methods and results In this study, we determined the ability of eDNA metabarcoding surveys to distinguish vertically localized community assemblages. To test this, we sampled three vertical transects along a steep rock wall at three depths (0 m, 4 m, 15 m), covering two distinct communities that were separated by near‐permanent water column stratification in the form of a strong halocline at ~3 m. Using three metabarcoding assays, our eDNA metabarcoding survey detected 54 taxa, across 46 families and 7 phyla, including 19 fish, 15 crustacean, and 8 echinoderm species. Ordination and cluster analyses show distinct eDNA signals across the halocline for all three replicate transects, suggesting that vertical dispersal of eDNA between communities was limited. Furthermore, eDNA signals of individual taxa were only retrieved within their observed vertical distribution, providing biological validation for the obtained results. Our results demonstrate, for the first time, the need to take into consideration oceanographic (e.g. water column stratification) and biological processes (e.g. vertical community structuring) when designing sampling strategies for marine eDNA metabarcoding surveys.

The age, sex, and sexual maturity of individual animals are key parameters in assessing wild populations and informing conservation management strategies. These parameters represent the reproductive potential of a population and can indicate recovery rates or vulnerabilities. Natural populations of wild animals are difficult to study; logistically, economically, and due to the impacts of invasive biomonitoring. Genetic and epigenetic analyses offer a low impact, low cost, and information-rich alternative. As epigenetic mechanisms are intrinsically linked with both biological aging and reproductive processes, DNA methylation can be used as a suitable biomarker for population biology study. This review assesses published research utilizing DNA methylation analysis in relation to three key population parameters: age, sex, and sexual maturity. We review studies on wild vertebrates that investigate epigenetic age relationships, with successful age estimation assays designed for mammals, birds, and fish. For both determination of sex and identification of sexual maturity, very little has been explored regarding DNA methylation-based assays. Related research, however, confirms the links between DNA methylation and these processes. Future development of age estimation assays for underrepresented and key conservation taxa is suggested, as is the experimental development and design of DNA methylation-based assays for both sex and sexual maturity identification, further expanding the genomics toolkit for population biology studies.

Decommissioning of offshore oil and gas structures is either occurring, or imminent in most regions of the world. Most jurisdictions require that offshore structures be removed for onshore disposal. However, there is growing interest in understanding the ecological and socio-economic benefits of leaving structures in the water. Descriptions of how fish utilize the vertical structure created by wellhead platform jackets (platforms) will provide insights into possible outcomes of decommissioning alternatives, such as full removal, leave in situ, or translocation to a designated reefing site. We surveyed fish assemblages associated with seven platforms and five reference sites located ∼150 km offshore in the central Gulf of Thailand. The platforms spanned the entire water column (∼75 m) and were a mix of three and four legged structures. We used a Remotely Operated Vehicle (ROV) fitted with an underwater stereo video system to quantify the abundance, size, biomass, and economic value of fish associated with the platforms. We recorded 43 species of fish on the platforms and five reference sites with most fishes on platforms categorized as coral-reef or coral-reef-associated species. We observed a strong vertical zonation in the fish assemblage on the platforms. The Regal demoiselle (Neopomacentrus cyanomos) was numerically dominant (75% of all fish observed). We measured 3,933 kg of fish on the platforms with Caranx sexfasciatus accounting for 76.12% of that. We conservatively estimate each platform had a scaled mean biomass of ∼2,927 kg and the fished species had scaled mean economic value of 175,500 Thai Baht per platform. We estimated that the biomass of fish associated with the seven platforms was at least four times higher per unit area than some of the world’s most productive coral reefs.

The role of fungi in the biogeochemical cycling of gold remains unclear. Here the authors show that fungi can initiate gold oxidation under supergene conditions, thereby impacting gold mobilisation and secondary deposit formation in terrestrial environments.

Abstract DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.

Though there are multiple viable powertrain options available for the automotive sector, those that contain internal combustion engines will continue to account for the majority of global sales for the next several decades. It is therefore imperative to continue the pursuit of novel combustion concepts that produce efficiency levels significantly higher than those of current engines. Introducing high levels of dilution in spark ignited (SI) engines has consistently proven to produce an efficiency benefit compared to conventional stoichiometric engine operation. However, this combustion mode can present challenges for the ignition system. Pre-chamber jet ignition enables stable, highly dilute combustion by both increasing the ignition energy present in the system and distributing it throughout the combustion chamber. Previous work by the authors have shown that jet ignition produces 15–25% increases in thermal efficiency over baseline SI engines with only relatively minor changes to engine architecture. Lean combustion in general and jet ignition in particular represent fundamentally different engine operating modes compared to those of conventional stoichiometric SI engines. Therefore, there are some system sensitivities not present in stoichiometric engines that must be investigated in order to fully optimize the jet ignition system. Differing types and magnitudes of charge motion are incorporated in SI engines to aid with mixture preparation but the influence of charge motion over lean combustion performance, particularly in jet ignition engines, is less well understood. This study analyzes the impact that charge motion has on both pre-chamber and main chamber combustion. A 1.5 L 3-cylinder gasoline engine is outfitted with multiple intake port configurations producing varying magnitudes and types of charge motion. Pre-chamber and main chamber combustion stability and other burn parameter responses are analyzed across multiple speeds and loads, including at critical operating points such as a catalyst heating condition. The results show that there is combustion sensitivity to charge motion, resulting in >1 percentage point spread in peak thermal efficiency for the configurations tested, and that this sensitivity manifests most significantly under low ignitability conditions such as heavy dilution. These results provide guidance for future system optimization of jet ignition engines.

In the absence of a primary crop host, secondary plant hosts may act as a reservoir for fungal plant pathogens of agricultural crops. Secondary hosts may potentially harbor heteroecious biotrophs (e.g., the stripe rust fungus Puccinia striiformis) or other pathogens with broad host ranges. Agricultural grain production tends toward monoculture or a limited number of crop hosts over large regions, and local weeds are a major source of potential secondary hosts. In this study, the fungal phyllospheres of 12 weed species common in the agricultural regions of Western Australia (WA) were compared through high-throughput DNA sequencing. Amplicons of D2 and ITS were sequenced on an Illumina MiSeq system using previously published primers and BLAST outputs analyzed using MEGAN. A heatmap of cumulative presence–absence for fungal taxa was generated, and variance patterns were investigated using principal components analysis (PCA) and canonical correspondence analysis (CCA). We observed the presence of several major international crop pathogens, including basidiomycete rusts of the Puccinia spp., and ascomycete phytopathogens of the Leptosphaeria and Pyrenophora genera. Unrelated to crop production, several endemic pathogen species including those infecting Eucalyptus trees were also observed, which was consistent with local native flora. We also observed that differences in latitude or climate zones appeared to influence the geographic distributions of plant pathogenic species more than the presence of compatible host species, with the exception of Brassicaceae host family. There was an increased proportion of necrotrophic Ascomycete species in warmer and drier regions of central WA, compared to an increased proportion of biotrophic Basidiomycete species in cooler and wetter regions in southern WA.

Remotely operated vehicles (ROVs) are used extensively by the offshore oil and gas and renewables industries for inspection, maintenance, and repair of their infrastructure. With thousands of subsea structures monitored across the world’s oceans from the shallows to depths greater than 1,000 m, there is a great and underutilized opportunity for their scientific use. Through slight modifications of ROV operations, and by augmenting industry workclass ROVs with a range of scientific equipment, industry can fuel scientific discoveries, contribute to an understanding of the impact of artificial structures in our oceans, and collect biotic and abiotic data to support our understanding of how oceans and marine life are changing. Here, we identify and describe operationally feasible methods to adjust the way in which industry ROVs are operated to enhance the scientific value of data that they collect, without significantly impacting scheduling or adding to deployment costs. These include: rapid marine life survey protocols, imaging improvements, the addition of a range of scientific sensors, and collection of biological samples. By partnering with qualified and experienced research scientists, industry can improve the quality of their ROV-derived data, allowing the data to be analyzed robustly. Small changes by industry now could provide substantial benefits to scientific research in the long-term and improve the quality of scientific data in existence once the structures require decommissioning. Such changes also have the potential to enhance industry’s environmental stewardship by improving their environmental management and facilitating more informed engagement with a range of external stakeholders, including regulators and the public.

Bacterial communities are fundamental symbionts of corals. However, the process by which bacterial communities are acquired across the life history of corals, particularly in larval and early juvenile stages, is still poorly characterized. Here, transfer of bacteria of the Scleractinian coral Acropora digitifera from adults to spawned egg-sperm bundles was analyzed, as well as acquisition across early developmental stages (larvae and newly settled spat), and 6-month-old juveniles. Larvae were reared under manipulated environmental conditions to determine the source (maternal, seawater, or sediment) of bacteria likely to establish symbiotic relationships with the host using amplicon sequencing of the 16S rRNA gene. Maternal colonies directly transferred bacteria from the families Rhodobacteraceae, Cryomorphaceae, and Endozoicimonaceae to egg-sperm bundles. Furthermore, significant differences in the microbial community structure were identified across generations, yet the structure of the coral bacterial community across early life history stages was not impacted by different environmental rearing conditions. These data indicate that the uptake and structure of bacterial communities is developmentally, rather than environmentally, regulated. Both maternal coral colonies and ubiquitous bacteria found across environmental substrates represent a potential source of symbionts important in establishing the coral microbiome. Uniquely, we report the presence of variation with ontogeny of both the core and resident bacterial communities, supporting the hypothesis that microbial communities are likely to play specific roles within the distinct life history stages of the coral host.

Marine ecosystems are changing rapidly as the oceans warm and become more acidic. The physical factors and the changes to ocean chemistry that they drive can all be measured with great precision. Changes in the biological composition of communities in different ocean regions are far more challenging to measure because most biological monitoring methods focus on a limited taxonomic or size range. Environmental DNA (eDNA) analysis has the potential to solve this problem in biological oceanography, as it is capable of identifying a huge phylogenetic range of organisms to species level. Here we develop and apply a novel multi-gene molecular toolkit to eDNA isolated from bulk plankton samples collected over a five-year period from a single site. This temporal scale and level of detail is unprecedented in eDNA studies. We identified consistent seasonal assemblages of zooplankton species, which demonstrates the ability of our toolkit to audit community composition. We were also able to detect clear departures from the regular seasonal patterns that occurred during an extreme marine heatwave. The integration of eDNA analyses with existing biotic and abiotic surveys delivers a powerful new long-term approach to monitoring the health of our world's oceans in the context of a rapidly changing climate.

Background Effective biodiversity monitoring is fundamental in tracking changes in ecosystems as it relates to commercial, recreational, and conservation interests. Current approaches to survey coral reef ecosystems center on the use of indicator species and repeat surveying at specific sites. However, such approaches are often limited by the narrow snapshot of total marine biodiversity that they describe and are thus hindered in their ability to contribute to holistic ecosystem-based monitoring. In tandem, environmental DNA (eDNA) and next-generation sequencing metabarcoding methods provide a new opportunity to rapidly assess the presence of a broad spectrum of eukaryotic organisms within our oceans, ranging from microbes to macrofauna. Methods We here investigate the potential for rapid universal metabarcoding surveys (RUMS) of eDNA in sediment samples to provide snapshots of eukaryotic subtropical biodiversity along a depth gradient at two coral reefs in Okinawa, Japan based on 18S rRNA. Results Using 18S rRNA metabarcoding, we found that there were significant separations in eukaryotic community assemblages (at the family level) detected in sediments when compared across different depths ranging from 10 to 40 m (p = 0.001). Significant depth zonation was observed across operational taxonomic units assigned to the class Demospongiae (sponges), the most diverse class (contributing 81% of species) within the phylum Porifera; the oldest metazoan phylum on the planet. However, zonation was not observed across the class Anthozoa (i.e., anemones, stony corals, soft corals, and octocorals), suggesting that the former may serve as a better source of indicator species based on sampling over fine spatial scales and using this universal assay. Furthermore, despite their abundance on the examined coral reefs, we did not detect any octocoral DNA, which may be due to low cellular shedding rates, assay sensitivities, or primer biases. Discussion Overall, our pilot study demonstrates the importance of exploring depth effects in eDNA and suggest that RUMS may be applied to provide a baseline of information on eukaryotic marine taxa at coastal sites of economic and conservation importance.

Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler’s encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.

Globally, marine species’ distributions are being modified due to rising ocean temperatures. Increasing evidence suggests a circum-global pattern of poleward extensions in the distributions of many tropical herbivorous species, including the ecologically important rabbitfish Siganus fuscescens. Adaptability of a species to such new environments may be heavily influenced by the composition of their gastrointestinal microbe fauna, which is fundamentally important to animal health. Siganus fuscescens thus provides an opportunity to assess the stability of gastrointestinal microbes under varying environmental conditions. The gastrointestinal microbial communities of S. fuscescens were characterized over 2,000 km of Australia’s western coast, from tropical to temperate waters, including near its current southern distributional limit. Sequencing of the 16S rRNA gene demonstrated that each population had a distinct hindgut microbial community, and yet, 20 OTUs occurred consistently in all samples. These OTUs were considered the ‘core microbiome’ and were highly abundant, composing between 31 and 54% of each population. Furthermore, levels of short chain fatty acids, an indicator of microbial fermentation activity, were similar among tropical and temperate locations. These data suggest that flexibility in the hindgut microbiome may play a role in enabling such herbivores to colonize new environments beyond their existing range.

Abstract Background The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks. Methods Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM. Results Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai. Conclusions Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel “Candidatus Neoehrlichia” spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

The recently extinct New Zealand adzebills (Aptornithidae, Aptornis spp.) were an enigmatic group of large flightless birds that have long eluded precise taxonomic assignment as they do not closely resemble any extant birds. Adzebills were nearly wingless, weighed approximately 16⁻19 kg, and possessed massive adze-like reinforced bills whose function remains unknown. Using hybridisation enrichment and high-throughput sequencing of DNA extracted from subfossil bone and eggshell, near-complete mitochondrial genomes were successfully assembled from the two Quaternary adzebill species: the North Island Adzebill (Aptornis otidiformis) and South Island Adzebill (A. defossor). Molecular phylogenetic analyses confirm that adzebills are members of the Ralloidea (rails and allies) and are sister-taxon to the Sarothruridae, which our results suggest comprises the Madagascan wood rails (Mentocrex, two likely sp.) in addition to the tiny (Sarothrura, 9 spp.). Node age estimates indicate that the split between adzebills and Sarothruridae occurred ~39.6 Ma, suggesting that the ancestors of the adzebills arrived in New Zealand by long-distance dispersal rather than continental vicariance. This newly identified biogeographic link between physically distant New Zealand and Afro-Madagascar, echoed by the relationship between the New Zealand kiwi (Apterygiformes) and Madagascan elephant-birds (Aepyornithiformes), suggests that the adzebill’s near relatives were formerly more widespread. In addition, our estimate for the divergence time between the two Quaternary adzebill species (0.2⁻2.3 Ma) coincides with the emergence of a land-bridge between the North and South islands of New Zealand (ca. 1.5⁻2 Ma). This relatively recent divergence suggests that North Island adzebills are the result of a relatively recent dispersal from the South Island, from which the earliest (Miocene) adzebill fossil has been described.

Genetic variation in microsatellites is rarely examined in the field of ancient DNA (aDNA) due to the low quantity of nuclear DNA in the fossil record together with the lack of characterized nuclear markers in extinct species. 454 sequencing platforms provide a new high-throughput technology capable of generating up to 1 gigabases per run as short (200–400-bp) read lengths. 454 data were generated from the fossil bone of an extinct New Zealand moa (Aves: Dinornithiformes). We identified numerous short tandem repeat (STR) motifs, and here present the successful isolation and characterization of one polymorphic microsatellite (Moa_MS2). Primers designed to flank this locus amplified all three moa species tested here. The presented method proved to be a fast and efficient way of identifying microsatellite markers in ancient DNA templates and, depending on biomolecule preservation, has the potential of enabling high-resolution population genetic studies of extinct taxa. As sequence read lengths of the 454 platforms and its competitors (e.g., the SOLEXA and SOLiD platforms) increase, this approach will become increasingly powerful in identifying microsatellites in extinct (and extant) organisms, and will afford new opportunities to study past biodiversity and extinction processes.

Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose.

The marsupial genus Macropus includes three subgenera, the familiar large grazing kangaroos and wallaroos of M. (Macropus) and M. (Osphranter), as well as the smaller mixed grazing/browsing wallabies of M. (Notamacropus). A recent study of five concatenated nuclear genes recommended subsuming the predominantly browsing Wallabia bicolor (swamp wallaby) into Macropus. To further examine this proposal we sequenced partial mitochondrial genomes for kangaroos and wallabies. These sequences strongly favour the morphological placement of W. bicolor as sister to Macropus, although place M. irma (black-gloved wallaby) within M. (Osphranter) rather than as expected, with M. (Notamacropus). Species tree estimation from separately analysed mitochondrial and nuclear genes favours retaining Macropus and Wallabia as separate genera. A simulation study finds that incomplete lineage sorting among nuclear genes is a plausible explanation for incongruence with the mitochondrial placement of W. bicolor, while mitochondrial introgression from a wallaroo into M. irma is the deepest such event identified in marsupials. Similar such coalescent simulations for interpreting gene tree conflicts will increase in both relevance and statistical power as species-level phylogenetics enters the genomic age. Ecological considerations in turn, hint at a role for selection in accelerating the fixation of introgressed or incompletely sorted loci. More generally the inclusion of the mitochondrial sequences substantially enhanced phylogenetic resolution. However, we caution that the evolutionary dynamics that enhance mitochondria as speciation indicators in the presence of incomplete lineage sorting may also render them especially susceptible to introgression.

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when plant reference databases become better established.

We present the first set of microsatellite markers developed exclusively for an extinct taxon. Microsatellite data have been analysed in thousands of genetic studies on extant species but the technology can be problematic when applied to low copy number (LCN) DNA. It is therefore rarely used on substrates more than a few decades old. Now, with the primers and protocols presented here, microsatellite markers are available to study the extinct New Zealand moa (Aves: Dinornithiformes) and, as with single nucleotide polymorphism (SNP) technology, the markers represent a means by which the field of ancient DNA can (preservation allowing) move on from its reliance on mitochondrial DNA. Candidate markers were identified using high throughput sequencing technology (GS-FLX) on DNA extracted from fossil moa bone and eggshell. From the 'shotgun' reads, >60 primer pairs were designed and tested on DNA from bones of the South Island giant moa (Dinornis robustus). Six polymorphic loci were characterised and used to assess measures of genetic diversity. Because of low template numbers, typical of ancient DNA, allelic dropout was observed in 36-70% of the PCR reactions at each microsatellite marker. However, a comprehensive survey of allelic dropout, combined with supporting quantitative PCR data, allowed us to establish a set of criteria that maximised data fidelity. Finally, we demonstrated the viability of the primers and the protocols, by compiling a full Dinornis microsatellite dataset representing fossils of c. 600-5000 years of age. A multi-locus genotype was obtained from 74 individuals (84% success rate), and the data showed no signs of being compromised by allelic dropout. The methodology presented here provides a framework by which to generate and evaluate microsatellite data from samples of much greater antiquity than attempted before, and opens new opportunities for ancient DNA research.

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.

Prior to human settlement 700 years ago New Zealand had no terrestrial mammals--apart from three species of bats--instead, approximately 250 avian species dominated the ecosystem. At the top of the food chain was the extinct Haast's eagle, Harpagornis moorei. H. moorei (10-15 kg; 2-3 m wingspan) was 30%-40% heavier than the largest extant eagle (the harpy eagle, Harpia harpyja), and hunted moa up to 15 times its weight. In a dramatic example of morphological plasticity and rapid size increase, we show that the H. moorei was very closely related to one of the world's smallest extant eagles, which is one-tenth its mass. This spectacular evolutionary change illustrates the potential speed of size alteration within lineages of vertebrates, especially in island ecosystems.