Analysing the effects of distance, taxon and biomass on vertebrate detections using bulk-collected carrion fly iDNA.

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.

TICI: a taxon-independent community index for eDNA-based ecological health assessment.

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.

A large scale temporal and spatial environmental DNA biodiversity survey of marine vertebrates in Brazil following the Fundão tailings dam failure.

Seawater contains a wealth of genetic information, representing the biodiversity of numerous species residing within a particular marine habitat. Environmental DNA (eDNA) metabarcoding offers a cost effective, non-destructive method for large scale monitoring of environments, as diverse taxonomic groups are detected using metabarcoding assays. A large-scale eDNA monitoring program of marine vertebrates was conducted across three sampling seasons (Spring 2018, Autumn 2019; Spring 2019) in coastal waters of Brazil. The program was designed to investigate eDNA as a testing method for long term monitoring of marine vertebrates following the Fundão tailings dam failure in November 2015. While no baseline samples were available prior to the dam failure there is still value in profiling the taxa that use the impacted area and the trajectory of recovery. A total of 40 sites were sampled around the mouths of eight river systems, covering approximately 500 km of coastline. Metabarcoding assays targeting the mitochondrial genes 16S rRNA and COI were used to detect fish, marine mammals and elasmobranchs. We detected temporal differences between seasons and spatial differences between rivers/estuaries sampled. Overall, the largest eDNA survey in Brazil to date revealed 69 families from Class Actinopterygii (fish), 15 species from Class Chondrichthyes (sharks and rays), 4 species of marine and estuarine mammals and 23 species of conservation significance including 2 species of endangered dolphin. Our large-scale study reinforces the value eDNA metabarcoding can bring when monitoring the biodiversity of coastal environments and demonstrates the importance of collection of time-stamped environmental samples to better understand the impacts of anthropogenic activities.

Gut content metabarcoding of specialized feeders is not a replacement for environmental DNA assays of seawater in reef environments.

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.

Plumbing the depths with environmental DNA (eDNA): Metabarcoding reveals biodiversity zonation at 45-60 m on mesophotic coral reefs.

Mesophotic coral ecosystems (MCEs) are tropical reefs found at depths of ~30-150 m, below the region most heavily impacted by heat stress and other disturbances. Hence, MCEs may serve as potential refugia for threatened shallow reefs, but they also harbour depth-endemic fauna distinct from shallow reefs. Previous studies have characterized biodiversity patterns along depth gradients, but focussed primarily on conspicuous taxa (fishes, corals, etc.). Environmental DNA (eDNA) metabarcoding offers a more holistic approach to assess biodiversity patterns across the tree of life. Here, we use three metabarcoding assays targeting fishes (16S rRNA), eukaryotes (18S rDNA) and metazoans (COI) to assess biodiversity change from the surface to ~90 m depth across 15-m intervals at three sites within the Hawaiian Archipelago. We observed significant community differences between most depth zones, with distinct zonation centred at 45-60 m for eukaryotes and metazoans, but not for fishes. This finding may be attributable to the higher mobility of reef fishes, although methodological limitations are likely a contributing factor. The possibility for MCEs to serve as refugia is not excluded for fishes, but invertebrate communities >45 m are distinct, indicating limited connectivity for the majority of reef fauna. This study provides a new approach for surveying biodiversity on MCEs, revealing patterns in a much broader context than the limited-taxon studies that comprise the bulk of our present knowledge.

Using environmental DNA to better inform decision making around decommissioning alternatives for offshore oil and gas infrastructure.

Artificial reefs are being utilised globally to aid in natural resource management, conservation, restoration or the creation of unique marine habitats. There is discussion around the optimal construction materials and designs for artificial reefs, the influences these have on biological communities, and the resulting ecological and social benefits. This discussion also includes the ecological value of repurposed marine infrastructure, such as decommissioned oil and gas platforms. Platforms often have an operational life spanning multiple decades, over which time they can develop extensive and unique community assemblages. The creation of artificial reefs by repurposing oil and gas platforms can have ecological, economic and sociological merit. However, with >12,000 platforms requiring decommissioning globally, there is the need for holistic assessment of biological communities associated with these platforms to inform the potential outcomes of different decommissioning options. We use environmental DNA metabarcoding (eDNA) of water, bio-foul and sediment samples to census broad eukaryotic diversity at eight platforms in the Gulf of Thailand (GoT) and five nearby soft sediment habitat locations. We sampled three target depths at sites (shallow, mid, deep) and detected 430 taxa at platforms, with higher diversity in shallow (near-surface) samples (313 taxa), compared to mid (30 m collection depth; 261 taxa) and deep (50 m; 273 taxa). Three percent of taxa were shared among all depths at platforms with distinct assembles at each depth. Introduced species are an ongoing risk for platforms, however the eDNA detected no known introduced species. While the eDNA data provide broad taxon coverage and significant assemblage patterns, ongoing sampling innovation, assay design and local reference material still require development to obtain the maximum benefit of the technique. This study highlights the versatility and scalability of eDNA metabarcoding to holistically census marine infrastructure and inform the management and potential conservation of extant communities.

Exploring the depth and breadth of the genomics toolbox during the COVID-19 pandemic: insights from Aotearoa New Zealand.

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.

Use of carrion fly iDNA metabarcoding to monitor invasive and native mammals.

Severely fragmented habitats increase the risk of extirpation of native mammal populations through isolation, increased edge effects, and predation. Therefore, monitoring the movement of mammal populations through anthropogenically altered landscapes can inform conservation. We used metabarcoding of invertebrate-derived DNA (iDNA) from carrion flies (Calliphoridae and Sarcophagidae) to track mammal populations in the wheat belt of southwestern Australia, where widespread clearing for agriculture has removed most of the native perennial vegetation and replaced it with an agricultural system. We investigated whether the localization of the iDNA signal reflected the predicted distribution of 4 native species-echidna (Tachyglossus aculeatus), numbat (Myrmecobius fasciatus), woylie (Bettongia penicillata), and chuditch (Dasyurus geoffroii)-and 2 non-native, invasive mammal species-fox (Vulpes vulpes) and feral cat (Felis catus). We collected bulk iDNA samples (n = 150 samples from 3428 carrion flies) at 3 time points from 3 conservation reserves and 35 road edges between them. We detected 14 of the 40 mammal species known from the region, including our target species. Most detections of target taxa were in conservation reserves. There were a few detections from road edges. We detected foxes and feral cats throughout the study area, including all conservation reserves. There was a significant difference between the diversity (F3, 98  = 5.91, p < 0.001) and composition (F3, 43  = 1.72, p < 0.01) of taxa detections on road edges and conservation reserves. Conservation reserves hosted more native biodiversity than road edges. Our results suggest that the signals from iDNA reflect the known distribution of target mammals in this region. The development of iDNA methods shows promise for future noninvasive monitoring of mammals. With further development, iDNA metabarcoding could inform decision-making related to conservation of endangered taxa, invasive species management, and impacts of habitat fragmentation.

Caracterización genética del ADNi de la mosca carroñera para monitorear mamíferos invasores y nativos Resumen Los hábitats con mucha fragmentación aumentan el riesgo de extirpación de las poblaciones de mamíferos nativos debido al aislamiento, el aumento de los efectos de borde y la depredación. Por lo tanto, el monitoreo del movimiento de las poblaciones de mamíferos a través de paisajes alterados antropogénicamente puede guiar a la conservación. Utilizamos la caracterización genética del ADN derivado de invertebrados (ADNi) de moscas de la carroña (Calliphoridae y Sarcophagidae) para rastrear poblaciones de mamíferos en la región de Wheatbelt del suroeste de Australia, en donde la tala generalizada ha sustituido la mayor parte de la vegetación perenne nativa por un sistema agrícola. Investigamos si la localización de la señal de ADNi reflejaba la distribución prevista de cuatro especies autóctonas: equidna (Tachyglossus aculeatus), numbat (Myrmecobius fasciatus), rata canguro (Bettongia penicillata) y cuol occidental (Dasyurus geoffroii), y dos especies de mamíferos invasores no autóctonos: el zorro (Vulpes vulpes) y el gato feral (Felis catus). Recogimos muestras masivas de ADNi (n = 150 muestras de 3,428 moscas de la carroña) en tres puntos temporales de tres reservas ecológicas y 35 bordes de carreteras entre ellas. Detectamos 14 de las 40 especies de mamíferos conocidas en la región, incluidas nuestras especies objetivo. La mayoría de las detecciones de los taxones objetivo se produjeron en las reservas ecológicas. Pocas detecciones ocurrieron en los bordes de las carreteras. Detectamos zorros y gatos ferales en toda la zona de estudio, incluidas todas las reservas ecológicas. Hubo una diferencia significativa entre la diversidad (F3, 98 = 5.91, p<0.001) y la composición (F3, 43 = 1.72, p<0.01) de los taxones detectados en los bordes de las carreteras y en las reservas ecológicas. Las reservas ecológicas albergaron más biodiversidad nativa que los bordes de las carreteras. Nuestros resultados sugieren que las señales de ADNi reflejan la distribución conocida de los mamíferos objetivo en esta región. El desarrollo de métodos de ADNi es prometedor para el futuro monitoreo no invasivo de mamíferos. Con un mayor desarrollo, la caracterización genética del ADNi podría servir de base para decidir sobre la conservación de taxones amenazados, la gestión de especies invasoras y los impactos de la fragmentación del hábitat.

Gut microbial communities of hybridising pygmy angelfishes reflect species boundaries.

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.

It is time for ancient DNA to sweat the small stuff.

When one thinks of the field of ancient DNA it conjures images of extinct megafauna, from mammoths and woolly rhinos, through to the giant, flightless elephant bird (but hopefully not dinosaurs - despite the pervasive idea of 'dino DNA' from Jurassic park). These taxa have fascinating evolutionary histories, and their extinction stories need to be told. At the other end of the vertebrate scale, however, is the often neglected 'small stuff' - lizards, frogs, and other herpetofauna. But here's the rub - extracting DNA from the bones of this 'small stuff' is not only difficult, it often destroys the sample. In this issue, Scarsbrook et al. (2023) describe a new way to study the ancient (or historical) DNA of small vertebrates that is minimally destructive. The authors use the method to reconstruct the dynamic evolutionary history of New Zealand geckos and make new insights into how remnant populations should be managed. This work provides some key insights into New Zealand geckos but also opens up opportunities of biomolecular research on the smallest of vouchered vertebrate samples held within museum collections.

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.

Ancient DNA provides insights into 4,000 years of resource economy across Greenland.

The success and failure of past cultures across the Arctic was tightly coupled to the ability of past peoples to exploit the full range of resources available to them. There is substantial evidence for the hunting of birds, caribou and seals in prehistoric Greenland. However, the extent to which these communities relied on fish and cetaceans is understudied because of taphonomic processes that affect how these taxa are presented in the archaeological record. To address this, we analyse DNA from bulk bone samples from 12 archaeological middens across Greenland covering the Palaeo-Inuit, Norse and Neo-Inuit culture. We identify an assemblage of 42 species, including nine fish species and five whale species, of which the bowhead whale (Balaena mysticetus) was the most commonly detected. Furthermore, we identify a new haplotype in caribou (Rangifer tarandus), suggesting the presence of a distinct lineage of (now extinct) dwarfed caribou in Greenland 3,000 years ago.

Tracing the international arrivals of SARS-CoV-2 Omicron variants after Aotearoa New Zealand reopened its border.

In the second quarter of 2022, there was a global surge of emergent SARS-CoV-2 lineages that had a distinct growth advantage over then-dominant Omicron BA.1 and BA.2 lineages. By generating 10,403 Omicron genomes, we show that Aotearoa New Zealand observed an influx of these immune-evasive variants (BA.2.12.1, BA.4, and BA.5) through the border. This is explained by the return to significant levels of international travel following the border's reopening in March 2022. We estimate one Omicron transmission event from the border to the community for every ~5,000 passenger arrivals at the current levels of travel and restriction. Although most of these introductions did not instigate any detected onward transmission, a small minority triggered large outbreaks. Genomic surveillance at the border provides a lens on the rate at which new variants might gain a foothold and trigger new waves of infection.

DNA metabarcoding identifies urban foraging patterns of oligolectic and polylectic cavity-nesting bees.

Urbanisation modifies natural landscapes resulting in built-up space that is covered by buildings or hard surfaces and managed green spaces that often substitute native plant species with exotics. Some native bee species have been able to adapt to urban environments, foraging and reproducing in these highly modified areas. However, little is known on how the foraging ecology of native bees is affected by urbanised environments, and whether impacts vary among species with different degrees of specialisation for pollen collection. Here, we aim to investigate the responses of native bee foraging behaviour to urbanisation, using DNA metabarcoding to identify the resources within nesting tubes. We targeted oligolectic (specialist) and polylectic (generalist) cavity-nesting bee species in residential gardens and remnant bushland habitats. We were able to identify 40 families, 50 genera, and 23 species of plants, including exotic species, from the contents of nesting tubes. Oligolectic bee species had higher diversity of plant pollen in their nesting tubes in residential gardens compared to bushland habitats, along with significantly different forage composition between the two habitats. This result implies a greater degree of forage flexibility for oligolectic bee species than previously thought. In contrast, the diversity and composition of plant forage in polylectic bee nesting tubes did not vary between the two habitat types. Our results suggest a complex response of cavity-nesting bees to urbanisation and support the need for additional research to understand how the shifts in foraging resources impact overall bee health.

Key factors to consider in the use of environmental DNA metabarcoding to monitor terrestrial ecological restoration.

Ecological restoration of terrestrial environments is a globally important process to combat the loss of biodiversity and ecosystem services. Holistic monitoring of restored biota and active management of restoration is necessary to improve restoration processes and outcomes, and provide evidence to stakeholders that targets are being achieved. Increasingly, environmental DNA (eDNA) metabarcoding is used as a restoration monitoring tool because it is able to generate biodiversity data rapidly, accurately, non-destructively, and reliably, on a wide breadth of organisms from soil microbes to mammals. The overall objective of this review is to discuss the key factors to consider in the use of environmental DNA for monitoring of restored terrestrial ecosystems, hopefully improving monitoring, and ultimately, restoration outcomes. We identified that the majority of eDNA based studies of ecosystem restoration are currently conducted in Europe, North America, and Australia, and that almost half of total studies were published in 2021-22. Soil was the most popular sample substrate, soil microbial communities the most targeted taxa, and forests the most studied ecosystem. We suggest there is no 'one size fits all' approach to restoration monitoring using eDNA, and discuss survey design. Factors to consider include substrate selection, sample collection and storage, assay selection, and data interpretation, all of which require careful planning to obtain reliable, and accurate information that can be used for restoration monitoring and decision making. We explore future directions for research and argue that eDNA metabarcoding can be a useful tool in the restoration monitoring 'toolkit', but requires informed application and greater accessibility to data by a wide spectrum of stakeholders.

Complementary molecular and visual sampling of fish on oil and gas platforms provides superior biodiversity characterisation.

Offshore oil and gas platforms have the potential to provide complex refugia for fish and benthic colonisers. We compare two methods of biodiversity assessment for fish and elasmobranchs at seven decommissioned oil and gas platforms as well as five sediment sites, located 5 km from platforms, in the Gulf of Thailand. Using surveys from stereo-video ROV transects, and data from Environmental DNA (eDNA) water-column samples, we detected fish and elasmobranch taxa from 39 families and 66 genera across both platform and sediment sites with eDNA, compared with 18 families and 29 genera by stereo-ROV with platforms yielding significantly greater species richness. This study demonstrates that the combination of stereo-video ROV and eDNA provide effective, non-extractive and complementary methods to enhance data capture. This approach sets new benchmarks for evaluating fish assemblages surrounding platforms and will enhance measurements of biota to inform decisions on the fate of oil/gas infrastructure.

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.

Phylogeography of recent Plesiastrea (Scleractinia: Plesiastreidae) based on an integrated taxonomic approach.

Scleractinian corals are a diverse group of ecologically important yet highly threatened marine invertebrates, which can be challenging to identify to the species level. An influx of molecular studies has transformed scleractinian systematics, highlighting that cryptic species may be more common than previously understood. In this study, we test the hypothesis that Plesiastrea versipora (Lamarck, 1816), a species currently considered to occur throughout the Indo-Pacific in tropical, sub-tropical and temperate waters, is a single species. Molecular and morphological analyses were conducted on 80 samples collected from 31 sites spanning the majority of the species putative range and twelve mitogenomes were assembled to identify informative regions for phylogenetic reconstruction. Congruent genetic data across three gene regions supports the existence of two monophyletic clades aligning with distinct tropical and temperate provenances. Multivariate macromorphological analyses based on 13 corallite characters provided additional support for the phylogeographic split, with the number of septa and corallite density varying across this biogeographic divide. Furthermore, micromorphological and microstructural analyses identified that the temperate representatives typically develop sub-cerioid corallites with sparse or absent coenosteal features and smooth septal faces. In contrast, tropical representatives typically develop plocoid corallites separated by a porous dissepimental coenosteum and have granulated septal faces. These data suggest that at least two species exist within the genus PlesiastreaMilne Edwards & Haime, 1848. Based on examination of type material, we retain the name Plesiastrea versipora (Lamarck, 1816) for the temperate representatives of the genus and resurrect the name Plesiastrea peroniMilne Edwards & Haime, 1857 for the tropical members. This study highlights how broadly distributed hard coral taxa still need careful re-examination through an integrated systematics approach to better understand their phylogeographic patterns. Furthermore, it demonstrates the utility of integrating micro-, macro-morphological and genetic datasets, and the importance of type specimens when dealing with taxonomic revisions of scleractinian taxa.

Evaluating restoration trajectories using DNA metabarcoding of ground-dwelling and airborne invertebrates and associated plant communities.

Invertebrates are important for restoration processes as they are key drivers of many landscape-scale ecosystem functions; including pollination, nutrient cycling and soil formation. However, invertebrates are often overlooked in restoration monitoring because they are highly diverse, poorly described, and time-consuming to survey, and require increasingly scarce taxonomic expertise to enable identification. DNA metabarcoding is a relatively new tool for rapid survey that is able to address some of these concerns, and provide information about the taxa with which invertebrates are interacting via food webs and habitat. Here, we evaluate how invertebrate communities may be used to determine ecosystem trajectories during restoration. We collected ground-dwelling and airborne invertebrates across chronosequences of mine-site restoration in three ecologically disparate locations in Western Australia and identified invertebrate and plant communities using DNA metabarcoding. Ground-dwelling invertebrates showed the clearest restoration signals, with communities becoming more similar to reference communities over time. These patterns were weaker in airborne invertebrates, which have higher dispersal abilities and therefore less local fidelity to environmental conditions. Although we detected directional changes in community composition indicative of invertebrate recovery, patterns observed were inconsistent between study locations. The inclusion of plant assays allowed identification of plant species, as well as potential food sources and habitat. We demonstrate that DNA metabarcoding of invertebrate communities can be used to evaluate restoration trajectories. Testing and incorporating new monitoring techniques such as DNA metabarcoding is critical to improving restoration outcomes.

Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.