An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding.

Anthropogenically forced changes in global freshwater biodiversity demand more efficient monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the appropriate spatio-temporal resolution of robust biodiversity assessment remains ambiguous. Here, using intensive, spatio-temporal eDNA sampling across space (five rivers in Europe and North America, with an upper range of 20-35 km between samples), time (19 timepoints between 2017 and 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers using eDNA. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of environmental conditions were taxon-specific, reflecting habitat filtering of communities rather than effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatio-temporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river community ecology during a time of environmental change.

Comparative population genomics of manta rays has global implications for management.

Understanding population connectivity and genetic diversity is of fundamental importance to conservation. However, in globally threatened marine megafauna, challenges remain due to their elusive nature and wide-ranging distributions. As overexploitation continues to threaten biodiversity across the globe, such knowledge gaps compromise both the suitability and effectiveness of management actions. Here, we use a comparative framework to investigate genetic differentiation and diversity of manta rays, one of the most iconic yet vulnerable groups of elasmobranchs on the planet. Despite their recent divergence, we show how oceanic manta rays (Mobula birostris) display significantly higher heterozygosity than reef manta rays (Mobula alfredi) and that M. birostris populations display higher connectivity worldwide. Through inferring modes of colonization, we reveal how both contemporary and historical forces have likely influenced these patterns, with important implications for population management. Our findings highlight the potential for fisheries to disrupt population dynamics at both local and global scales and therefore have direct relevance for international conservation of marine species.

Managing human-mediated range shifts: understanding spatial, temporal and genetic variation in marine non-native species.

The use of molecular tools to manage natural resources is increasingly common. However, DNA-based methods are seldom used to understand the spatial and temporal dynamics of species' range shifts. This is important when managing range shifting species such as non-native species (NNS), which can have negative impacts on biotic communities. Here, we investigated the ascidian NNS Ciona robusta, Clavelina lepadiformis, Microcosmus squamiger and Styela plicata using a combined methodological approach. We first conducted non-molecular biodiversity surveys for these NNS along the South African coastline, and compared the results with historical surveys. We detected no consistent change in range size across species, with some displaying range stability and others showing range shifts. We then sequenced a section of cytochrome c oxidase subunit I (COI) from tissue samples and found genetic differences along the coastline but no change over recent times. Finally, we found that environmental DNA metabarcoding data showed broad congruence with both the biodiversity survey and the COI datasets, but failed to capture the complete incidence of all NNS. Overall, we demonstrated how a combined methodological approach can effectively detect spatial and temporal variation in genetic composition and range size, which is key for managing both thriving NNS and threatened species. This article is part of the theme issue 'Species' ranges in the face of changing environments (part I)'.

Ecological changes have driven biotic exchanges across the Indian Ocean.

The Indian Ocean has a complex geological history that has drawn the attention of naturalists for almost a century now. Due to its tectonic history, many geological elements and processes have been evoked to explain the exchange of species between landmasses. Here, we revisited previous studies on twenty-three taxa to investigate trends across time since the Gondwana breakup. We investigated these datasets by applying a time-calibrated Bayesian framework to them and reconstructing their ancestral ranges. We conclude that ecological transformations have presented opportunities for the establishment of migrants. The role of donating and receiving migrants has shifted several times according to these transformations. Time-specific trends show weak evidence for the stepping-stones commonly suggested as physical routes between landmasses. However, before its collision with Asia, India may have served as an intermediary for such exchanges.

Secondary predation constrains DNA-based diet reconstruction in two threatened shark species.

Increasing fishing effort, including bycatch and discard practices, are impacting marine biodiversity, particularly among slow-to-reproduce taxa such as elasmobranchs, and specifically sharks. While some fisheries involving sharks are sustainably managed, collateral mortalities continue, contributing towards > 35% of species being threatened with extinction. To effectively manage shark stocks, life-history information, including resource use and feeding ecologies is pivotal, especially among those species with wide-ranging distributions. Two cosmopolitan sharks bycaught off eastern Australia are the common blacktip shark (Carcharhinus limbatus; globally classified as Near Threatened) and great hammerhead (Sphyrna mokarran; Critically Endangered). We opportunistically sampled the digestive tracts of these two species (and also any whole prey; termed the 'Russian-doll' approach), caught in bather-protection gillnets off northern New South Wales, to investigate the capacity for DNA metabarcoding to simultaneously determine predator and prey regional feeding ecologies. While sample sizes were small, S. mokkaran fed predominantly on stingrays and skates (Myliobatiformes and Rajiformes), but also teleosts, while C. limbatus mostly consumed teleosts. Metabarcoding assays showed extensive intermixing of taxa from the digestive tracts of predators and their whole prey, likely via the predator's stomach chyme, negating the opportunity to distinguish between primary and secondary predation. This Russian-doll effect requires further investigation in DNA metabarcoding studies focussing on dietary preferences and implies that any outcomes will need to be interpreted concomitant with traditional visual approaches.

Environmental DNA provides higher resolution assessment of riverine biodiversity and ecosystem function via spatio-temporal nestedness and turnover partitioning.

Rapidly assessing biodiversity is essential for environmental monitoring; however, traditional approaches are limited in the scope needed for most ecological systems. Environmental DNA (eDNA) based assessment offers enhanced scope for assessing biodiversity, while also increasing sampling efficiency and reducing processing time, compared to traditional methods. Here we investigated the effects of landuse and seasonality on headwater community richness and functional diversity, via spatio-temporal dynamics, using both eDNA and traditional sampling. We found that eDNA provided greater resolution in assessing biodiversity dynamics in time and space, compared to traditional sampling. Community richness was seasonally linked, peaking in spring and summer, with temporal turnover having a greater effect on community composition compared to localized nestedness. Overall, our assessment of ecosystem function shows that community formation is driven by regional resource availability, implying regional management requirements should be considered. Our findings show that eDNA based ecological assessment is a powerful, rapid and effective assessment strategy that enables complex spatio-temporal studies of community diversity and ecosystem function, previously infeasible using traditional methods.

Animals, protists and bacteria share marine biogeographic patterns.

Over millennia, ecological and evolutionary mechanisms have shaped macroecological patterns across the tree of life. Research describing these patterns at both regional and global scales has traditionally focused on the study of metazoan species. Consequently, there is a limited understanding of cross-phylum biogeographic structuring and an escalating need to understand the macroecology of both microscopic and macroscopic organisms. Here we used environmental DNA (eDNA) metabarcoding to explore the biodiversity of marine metazoans, protists and bacteria along an extensive and highly heterogeneous coastline. Our results showed remarkably consistent biogeographic structure across the kingdoms of life despite billions of years of evolution. Analyses investigating the drivers of these patterns for each taxonomic kingdom found that environmental conditions (such as temperature) and, to a lesser extent, anthropogenic stressors (such as fishing pressure and pollution) explained some of the observed variation. Additionally, metazoans displayed biogeographic patterns that suggested regional biotic homogenization. Against the backdrop of global pervasive anthropogenic environmental change, our work highlights the importance of considering multiple domains of life to understand the maintenance and drivers of biodiversity patterns across broad taxonomic, ecological and geographical scales.

How mountains and elevations shape the spatial distribution of beetles in Peninsular Malaysia.

This study was conducted to assess the spatial distribution of beetles in mountain ecosystems and their elevational diversity. Malaise, pitfall and light traps were used to collect beetles from nine different mountains in Malaysia from September 2014 to September 2016, where from Gunung Angsi, Gunung Belumut, Gunung Basor and Gunung Tebu samples were collected at 500 m and 1000 m (above sea level) elevations, while beetles were sampled at 500 m, 1000 m and 1500 masl from Gunung Benom, Gunung Inas, Cameron Highland, Gunung Besar Hantu and Gunung Basor. In this study, 9628 beetles belonging to 879 different species were collected with highest representation from family Staphylinidae and Carabidae. Chamah Highland had the highest beetle diversity followed by Gunung Benom, Gunung Inas, Cameron Highland, Gunung Belumut, and Gunung Basor. Chamah Highland was different to all mountains on abundance and species richness. The highest species richness was observed at 1000 m, followed by 500 m and 1500 m. We identified characteristic species associated with habitat conditions at Gunung Benoum and Gunung Inas mountains, according to INDVAL values. The beetle diversity of the sampled mountains showed multiple alpha and beta patterns according to type of mountain ecosystem and elevation, providing guidelines for the scientific community to underpin conservation efforts in Malaysia.

Phylogenomics and species delimitation for effective conservation of manta and devil rays.

Practical biodiversity conservation relies on delineation of biologically meaningful units. Manta and devil rays (Mobulidae) are threatened worldwide, yet morphological similarities and a succession of recent taxonomic changes impede the development of an effective conservation strategy. Here, we generate genome-wide single nucleotide polymorphism (SNP) data from a geographically and taxonomically representative set of manta and devil ray samples to reconstruct phylogenetic relationships and evaluate species boundaries under the general lineage concept. We show that nominal species units supported by alternative data sources constitute independently evolving lineages, and find robust evidence for a putative new species of manta ray in the Gulf of Mexico. Additionally, we uncover substantial incomplete lineage sorting indicating that rapid speciation together with standing variation in ancestral populations has driven phylogenetic uncertainty within Mobulidae. Finally, we detect cryptic diversity in geographically distinct populations, demonstrating that management below the species level may be warranted in certain species. Overall, our study provides a framework for molecular genetic species delimitation that is relevant to wide-ranging taxa of conservation concern, and highlights the potential for genomic data to support effective management, conservation and law enforcement strategies.

Evolutionary history of a Scottish harbour seal population.

Efforts to conserve marine mammals are often constrained by uncertainty over their population history. Here, we examine the evolutionary history of a harbour seal (Phoca vitulina) population in the Moray Firth, northeast Scotland using genetic tools and microsatellite markers to explore population change. Previous fine-scale analysis of UK harbour seal populations revealed three clusters in the UK, with a northeastern cluster that included our Moray Firth study population. Our analysis revealed that the Moray Firth cluster is an independent genetic group, with similar levels of genetic diversity across each of the localities sampled. These samples were used to assess historic abundance and demographic events in the Moray Firth population. Estimates of current genetic diversity and effective population size were low, but the results indicated that this population has remained at broadly similar levels following the population bottleneck that occurred after post-glacial recolonization of the area.

Executing multi-taxa eDNA ecological assessment via traditional metrics and interactive networks.

Current approaches to ecological assessment are limited by the traditional morpho-taxonomic methods presently employed and the inability to meet increasing demands for rapid assessments. Advancements in high throughput sequencing now enable rapid high-resolution ecological assessment using environmental DNA (eDNA). Here we test the ability of using eDNA-based ecological assessment methods against traditional assessment of two key indicator groups (diatoms and macroinvertebrates) and show how eDNA across multiple gene regions (COI, rbcL, 12S and 18S) can be used to infer interactive networks that link to ecological assessment criteria. We compared results between taxonomic and eDNA based assessments and found significant positive associations between macroinvertebrate (p < 0.001 R2 = 0.645) and diatom (p = 0.015, R2 = 0.222) assessment metrics. We further assessed the ability of eDNA based assessment to identify environmentally sensitive genera and found an order of magnitude greater potential for 18S, versus COI or rbcL, to determine environmental filtering of ecologically assessed communities. Lastly, we compared the ability of traditional metrics against co-occurrence network properties of our combined 18S, COI and rbcL indicator genera to infer habitat quality measures currently used by managers. We found that transitivity (network connectivity), linkage density and cohesion were significantly associated with habitat modification scores (HMS), whereas network properties were inconsistent with linking to the habitat quality score (HQS) metric. The incorporation of multi-marker eDNA network assessment opens up a means for finer scale ecological assessment, currently limited using traditional methods. While utilization of eDNA-based assessment is recommended, direct comparisons with traditional approaches are difficult as the methods are intrinsically different and should be treated as such with regards to future research. Overall, our findings show that eDNA can be used for effective ecological assessment while offering a wider range of scope and application compared to traditional assessment methods.

Detection of introduced and resident marine species using environmental DNA metabarcoding of sediment and water.

Environmental DNA (eDNA) surveys are increasingly being used for biodiversity monitoring, principally because they are sensitive and can provide high resolution community composition data. Despite considerable progress in recent years, eDNA studies examining how different environmental sample types can affect species detectability remain rare. Comparisons of environmental samples are especially important for providing best practice guidance on early detection and subsequent mitigation of non-indigenous species. Here we used eDNA metabarcoding of COI (cytochrome c oxidase subunit I) and 18S (nuclear small subunit ribosomal DNA) genes to compare community composition between sediment and water samples in artificial coastal sites across the United Kingdom. We first detected markedly different communities and a consistently greater number of distinct operational taxonomic units in sediment compared to water. We then compared our eDNA datasets with previously published rapid assessment biodiversity surveys and found excellent concordance among the different survey techniques. Finally, our eDNA surveys detected many non-indigenous species, including several newly introduced species, highlighting the utility of eDNA metabarcoding for both early detection and temporal / spatial monitoring of non-indigenous species. We conclude that careful consideration on environmental sample type is needed when conducting eDNA surveys, especially for studies assessing community change.

Metabarcoding for stomach-content analyses of Pygmy devil ray (Mobula kuhlii cf. eregoodootenkee): Comparing tissue and ethanol preservative-derived DNA.

The application of high-throughput sequencing to retrieve multi-taxon DNA from different substrates such as water, soil, and stomach contents has enabled species identification without prior knowledge of taxon compositions. Here we used three minibarcodes designed to target mitochondrial COI in plankton, 16S in fish, and 16S in crustaceans, to compare ethanol- and tissue-derived DNA extraction methodologies for metabarcoding. The stomach contents of pygmy devilrays (Mobula kuhlii cf. eregoodootenkee) were used to test whether ethanol-derived DNA would provide a suitable substrate for metabarcoding. The DNA barcoding assays indicated that tissue-derived operational taxonomic units (OTUs) were greater compared to those from extractions performed directly on the ethanol preservative. Tissue-derived DNA extraction is therefore recommended for broader taxonomic coverage. Metabarcoding applications should consider including the following: (i) multiple barcodes, both taxon specific (e.g., 12S or 16S) and more universal (e.g., COI or 18S) to overcome bias and taxon misidentification and (ii) PCR inhibitor removal steps that will likely enhance amplification yields. However, where tissue is limited or no longer available, but the ethanol-preservative medium is still available, metabarcoding directly from ethanol does recover the majority of common OTUs, suggesting the ethanol-retrieval method could be applicable for dietary studies. Metabarcoding directly from preservative ethanol may also be useful where tissue samples are limited or highly valued; bulk samples are collected, such as for rapid species inventories; or mixed-voucher sampling is conducted (e.g., for plankton, insects, and crustaceans).

Acidity promotes degradation of multi-species environmental DNA in lotic mesocosms.

Accurate quantification of biodiversity is fundamental to understanding ecosystem function and for environmental assessment. Molecular methods using environmental DNA (eDNA) offer a non-invasive, rapid, and cost-effective alternative to traditional biodiversity assessments, which require high levels of expertise. While eDNA analyses are increasingly being utilized, there remains considerable uncertainty regarding the dynamics of multispecies eDNA, especially in variable systems such as rivers. Here, we utilize four sets of upland stream mesocosms, across an acid-base gradient, to assess the temporal and environmental degradation of multispecies eDNA. Sampling included water column and biofilm sampling over time with eDNA quantified using qPCR. Our findings show that the persistence of lotic multispecies eDNA, sampled from water and biofilm, decays to non-detectable levels within 2 days and that acidic environments accelerate the degradation process. Collectively, the results provide the basis for a predictive framework for the relationship between lotic eDNA degradation dynamics in spatio-temporally dynamic river ecosystems.

Exploring hidden diversity in Southeast Asia's Dermogenys spp. (Beloniformes: Zenarchopteridae) through DNA barcoding.

Members of the freshwater halfbeak genus Dermogenys are hard to identify to the species level, despite several previous attempts to isolate fixed meristic, morphometric and colour pattern differences. This has led to ongoing confusion in scientific literature, records of species occurrence, and entries in museum collections. Here, a DNA barcoding study was conducted on the genus to gain further understanding of its taxonomic status across the Southeast Asian region. Fish were collected from 33 localities, spanning freshwater and brackish habitats in Malaysia, Western Indonesia, Thailand and Vietnam. In total, 290 samples of Dermogenys spp. were amplified for a 651 base pair fragment of the mitochondrial cytochrome oxidase c subunit I (COI) gene. Analysis was able to successfully differentiate the three species: D. collettei, D. siamensis, D. sumatrana; reveal the presence of a new putative species, Dermogenys sp., that was sampled in sympatry with D. collettei at three locations; as well as uncovering two genetic lineages of a fifth species, D. bispina, that display non-overlapping geographical distributions in drainages of northern Borneo; Kudat and Sandakan. This study expands the barcode library for Zenarchopteridae, demonstrates the efficacy of DNA barcoding techniques for differentiating Dermogenys species, and the potential thereof in species discovery.

Stepping stones to isolation: Impacts of a changing climate on the connectivity of fragmented fish populations.

In the marine environment, understanding the biophysical mechanisms that drive variability in larval dispersal and population connectivity is essential for estimating the potential impacts of climate change on the resilience and genetic structure of populations. Species whose populations are small, isolated and discontinuous in distribution will differ fundamentally in their response and resilience to environmental stress, compared with species that are broadly distributed, abundant and frequently exchange conspecifics. Here, we use an individual-based modelling approach, combined with a population genetics projection model, to consider the impacts of a warming climate on the population connectivity of two contrasting Antarctic fish species, Notothenia rossii and Champsocephalus gunnari. Focussing on the Scotia Sea region, sea surface temperatures are predicted to increase significantly by the end of the 21st century, resulting in reduced planktonic duration and increased egg and larval mortality. With shorter planktonic durations, the results of our study predict reduced dispersal of both species across the Scotia Sea, from Antarctic Peninsula sites to islands in the north and east, and increased dispersal among neighbouring sites, such as around the Antarctic Peninsula. Increased mortality modified the magnitude of population connectivity but had little effect on the overall patterns. Whilst the predicted changes in connectivity had little impact on the projected regional population genetic structure of N. rossii, which remained broadly genetically homogeneous within distances of ~1,500 km, the genetic isolation of C. gunnari populations in the northern Scotia Sea was predicted to increase with rising sea temperatures. Our study highlights the potential for increased isolation of island populations in a warming world, with implications for the resilience of populations and their ability to adapt to ongoing environmental change, a matter of high relevance to fisheries and ecosystem-level management.

Range-wide genomic data synthesis reveals transatlantic vicariance and secondary contact in Atlantic cod.

Recent advances in genetic and genomic analysis have greatly improved our understanding of spatial population structure in marine species. However, studies addressing phylogeographic patterns at oceanic spatial scales remain rare. In Atlantic cod (Gadus morhua), existing range-wide examinations suggest significant transatlantic divergence, although the fine-scale contemporary distribution of populations and potential for secondary contact are largely unresolved. Here, we explore transatlantic phylogeography in Atlantic cod using a data-synthesis approach, integrating multiple genome-wide single-nucleotide polymorphism (SNP) datasets representative of different regions to create a single range-wide dataset containing 1,494 individuals from 54 locations and genotyped at 796 common loci. Our analysis highlights significant transatlantic divergence and supports the hypothesis of westward post-glacial colonization of Greenland from the East Atlantic. Accordingly, our analysis suggests the presence of transatlantic secondary contact off eastern North America and supports existing perspectives on the phylogeographic history of Atlantic cod with an unprecedented combination of genetic and geographic resolution. Moreover, we demonstrate the utility of integrating distinct SNP databases of high comparability.

The complex evolutionary history and phylogeography of Caridina typus (Crustacea: Decapoda): long-distance dispersal and cryptic allopatric species.

The evolutionary history of the old, diverse freshwater shrimp genus Caridina is still poorly understood, despite its vast distribution - from Africa to Polynesia. Here, we used nuclear and mitochondrial DNA to infer the phylogeographic and evolutionary history of C. typus, which is one of only four species distributed across the entire range of the genus. Despite this species' potential for high levels of gene flow, questions have been raised regarding its phylogeographic structure and taxonomic status. We identified three distinct lineages that likely diverged in the Miocene. Molecular dating and ancestral range reconstructions are congruent with C. typus' early dispersal to Africa, possibly mediated by the Miocene Indian Ocean Equatorial Jet, followed by back dispersal to Australasia after the Jet's closure. Furthermore, several different species delimitation methods indicate each lineage represents a distinct (cryptic) species, contradicting current morphospecies delimitation of a single C. typus taxon. The evolutionary history of C. typus lineages is complex, in which ancient oceanic current systems and (currently unrecognised) speciation events preceded secondary sympatry of these cryptic species.

Plio-Pleistocene phylogeography of the Southeast Asian Blue Panchax killifish, Aplocheilus panchax.

The complex climatic and geological history of Southeast Asia has shaped this region's high biodiversity. In particular, sea level fluctuations associated with repeated glacial cycles during the Pleistocene both facilitated, and limited, connectivity between populations. In this study, we used data from two mitochondrial and three anonymous nuclear markers to determine whether a fresh/brackish water killifish, Aplocheilus panchax, Hamilton, 1822, could be used to further understand how climatic oscillations and associated sea level fluctuations have shaped the distribution of biota within this region, and whether such patterns show evidence of isolation within palaeodrainage basins. Our analyses revealed three major mitochondrial clades within A. panchax. The basal divergence of A. panchax mitochondrial lineages was approximately 3.5 Ma, whilst the subsequent divergence timings of these clades occurred early Pleistocene (~2.6 Ma), proceeding through the Pleistocene. Continuous phylogeographic analysis showed a clear west-east dispersal followed by rapid radiation across Southeast Asia. Individuals from Krabi, just north of the Isthmus of Kra, were more closely related to the Indian lineages, providing further evidence for a freshwater faunal disjunction at the Isthmus of Kra biogeographic barrier. Our results suggest that Sulawesi, across the Wallace Line, was colonised relatively recently (~30 ka). Nuclear DNA is less geographically structured, although Mantel tests indicated that nuclear genetic distances were correlated with geographic proximity. Overall, these results imply that recent gene flow, as opposed to historical isolation, has been the key factor determining patterns of nuclear genetic variation in A. panchax, however, some evidence of historical isolation is retained within the mitochondrial genome. Our study further validates the existence of a major biogeographic boundary at the Kra Isthmus, and also demonstrates the use of widely distributed fresh/brackishwater species in phylogeographic studies, and their ability to disperse across major marine barriers in relatively recent time periods.