Multi-method survey rediscovers critically endangered species and strengthens Madagascar's freshwater fish conservation.

Freshwater ecosystems are crucial for global biodiversity through supporting plant and animal species and providing essential resources. These ecosystems are under significant threat, particularly in island environments such as Madagascar. Our study focuses on the Amboaboa River basin, home to the rare and endemic fish species Rheocles derhami, last recorded in 2013. To assess the status of this and other threatened fish species including Ptychochromis insolitus and Paretroplus gymnopreopercularis, and to understand freshwater fish population dynamics in this biodiversity hotspot, we conducted a comprehensive survey using both environmental DNA (eDNA) and traditional fishing methods. While traditional methods effectively captured a diverse range of species, including several invasive aliens and the critically endangered endemic species that were the focus of this study, the eDNA approach detected only a fraction of these introduced species and struggled to identify some critically endangered endemics at the species level. This highlights the value of combining methods to enhance species detection. We also investigated the trade-offs associated with multi-primer assessments in eDNA analysis, focusing on three different primer combinations targeting the 12S mitochondrial gene: MiFish, Tele02, and Riaz. Additionally, we provided 12S reference barcodes for 10 species across 9 genera of fishes from the region to increase the coverage of the public reference databases. Overall, our study elucidates the current state of freshwater biodiversity in the Amboaboa River basin and underscores the value of employing multiple methods for effective conservation strategies.

Orchidinae-205: A new genome-wide custom bait set for studying the evolution, systematics, and trade of terrestrial orchids.

Terrestrial orchids are a group of genetically understudied, yet culturally and economically important plants. The Orchidinae tribe contains many species that produce edible tubers that are used for the production of traditional delicacies collectively called 'salep'. Overexploitation of wild orchids in the Eastern Mediterranean and Western Asia threatens to drive many of these species to extinction, but cost-effective tools for monitoring their trade are currently lacking. Here we present a custom bait kit for target enrichment and sequencing of 205 novel genetic markers that are tailored to phylogenomic applications in Orchidinae s.l. A subset of 31 markers capture genes putatively involved in the production of glucomannan, a water-soluble polysaccharide that gives salep its distinctive properties. We tested the kit on 73 taxa native to the area, demonstrating universally high locus recovery irrespective of species identity, that exceeds the total sequence length obtained with alternative kits currently available. Phylogenetic inference with concatenation and coalescent approaches was robust and showed high levels of support for most clades, including some which were previously unresolved. Resolution for hybridizing and recently radiated lineages remains difficult, but could be further improved by analysing multiple haplotypes and the non-exonic sequences captured by our kit, with the promise to shed new light on the evolution of enigmatic taxa with a complex speciation history. Offering a step-up from traditional barcoding and universal markers, the genome-wide custom loci targeted by Orchidinae-205 are a valuable new resource to study the evolution, systematics and trade of terrestrial orchids.

Harnessing eDNA metabarcoding to investigate fish community composition and its seasonal changes in the Oslo fjord.

In the face of global ecosystem changes driven by anthropogenic activities, effective biomonitoring strategies are crucial for mitigating impacts on vulnerable aquatic habitats. Time series analysis underscores a great significance in understanding the dynamic nature of marine ecosystems, especially amidst climate change disrupting established seasonal patterns. Focusing on Norway's Oslo fjord, our research utilises eDNA-based monitoring for temporal analysis of aquatic biodiversity during a one year period, with bi-monthly sampling along a transect. To increase the robustness of the study, a taxonomic assignment comparing BLAST+ and SINTAX approaches was done. Utilising MiFish and Elas02 primer sets, our study detected 63 unique fish species, including several commercially important species. Our findings reveal a substantial increase in read abundance during specific migratory cycles, highlighting the efficacy of eDNA metabarcoding for fish composition characterization. Seasonal dynamics for certain species exhibit clear patterns, emphasising the method's utility in unravelling ecological complexities. eDNA metabarcoding emerges as a cost-effective tool with considerable potential for fish community monitoring for conservation purposes in dynamic marine environments like the Oslo fjord, contributing valuable insights for informed management strategies.

Complementary authentication of Chinese herbal products to treat endometriosis using DNA metabarcoding and HPTLC shows a high level of variability.

Traditional Chinese Medicine (TCM) is popular for the treatment of endometriosis, a complex gynecological disease that affects 10% of women globally. The growing market for TCMs has yielded a significant incentive for product adulteration, and although emerging technologies show promise to improve their quality control, many challenges remain. We tested the authenticity of two traditional Chinese herbal formulae used in women's healthcare for the treatment of endometriosis, known as Gui Zhi Fu Ling Wan (FL) and Ge Xia Zhu Yu Tang (GX). Dual-locus DNA metabarcoding analysis coupled with high-performance thin-layer chromatography (HPTLC) were used to authenticate 19 FL and six GX commercial herbal products, as well as three ad hoc prepared artificial mixtures. HPTLC was able to detect most of the expected ingredients via comparative component analysis. DNA metabarcoding was able to detect an unexpected species diversity in the products, including 38 unexpected taxa. Chromatography has a resolution for all species indirectly through the identification of marker compounds for the different species ingredients. Metabarcoding on the other hand yields an overview of species diversity in each sample, but interpretation of the results can be challenging. Detected species might not be present in quantities that matter, and without validated quantification, some detected species can be hard to interpret. Comparative analysis of the two analytical approaches also reveals that DNA for species might be absent or too fragmented to amplify as the relevant chemical marker compounds can be detected but no amplicons are assigned to the same species. Our study emphasizes that integrating DNA metabarcoding with phytochemical analysis brings valuable data for the comprehensive authentication of Traditional Chinese Medicines ensuring their quality and safe use.

DNA Metabarcoding for Quality Control of Basil, Oregano, and Paprika.

Herbs and spices are some of the most vulnerable products in terms of fraud and adulteration in the food sector. Although standard analytical methods are accurate for quality control of specific lead or marker compounds, they cannot accurately assess the entire species composition of many marketed products. Complementary analytical approaches are thus often used for comprehensive screening of herbs and spices. In this study we evaluate DNA metabarcoding for the identification and authentication of 62 products, containing basil, oregano, and paprika collected from different retailers and importers in Norway. Our results show varying degrees of discrepancy between the constituent species and those listed on the product labels, despite high product authenticity. We suggest the false positives result from the sensitivity of DNA metabarcoding and filtering thresholds should be integrated into protocols to reduce false positives. Our results highlight how integrating DNA metabarcoding into the toolbox of analytical methods for quality control of fresh and/or processed plant-based food can improve product quality.

The genome of Draba nivalis shows signatures of adaptation to the extreme environmental stresses of the Arctic.

The Arctic is one of the most extreme terrestrial environments on the planet. Here, we present the first chromosome-scale genome assembly of a plant adapted to the high Arctic, Draba nivalis (Brassicaceae), an attractive model species for studying plant adaptation to the stresses imposed by this harsh environment. We used an iterative scaffolding strategy with data from short-reads, single-molecule long reads, proximity ligation data, and a genetic map to produce a 302 Mb assembly that is highly contiguous with 91.6% assembled into eight chromosomes (the base chromosome number). To identify candidate genes and gene families that may have facilitated adaptation to Arctic environmental stresses, we performed comparative genomic analyses with nine non-Arctic Brassicaceae species. We show that the D. nivalis genome contains expanded suites of genes associated with drought and cold stress (e.g., related to the maintenance of oxidation-reduction homeostasis, meiosis, and signaling pathways). The expansions of gene families associated with these functions appear to be driven in part by the activity of transposable elements. Tests of positive selection identify suites of candidate genes associated with meiosis and photoperiodism, as well as cold, drought, and oxidative stress responses. Our results reveal a multifaceted landscape of stress adaptation in the D. nivalis genome, offering avenues for the continued development of this species as an Arctic model plant.

Genomic evidence of speciation reversal in ravens.

Many species, including humans, have emerged via complex reticulate processes involving hybridisation. Under certain circumstances, hybridisation can cause distinct lineages to collapse into a single lineage with an admixed mosaic genome. Most known cases of such 'speciation reversal' or 'lineage fusion' involve recently diverged lineages and anthropogenic perturbation. Here, we show that in western North America, Common Ravens (Corvus corax) have admixed mosaic genomes formed by the fusion of non-sister lineages ('California' and 'Holarctic') that diverged ~1.5 million years ago. Phylogenomic analyses and concordant patterns of geographic structuring in mtDNA, genome-wide SNPs and nuclear introns demonstrate long-term admixture and random interbreeding between the non-sister lineages. In contrast, our genomic data support reproductive isolation between Common Ravens and Chihuahuan Ravens (C. cryptoleucus) despite extensive geographic overlap and a sister relationship between Chihuahuan Ravens and the California lineage. These data suggest that the Common Raven genome was formed by secondary lineage fusion and most likely represents a case of ancient speciation reversal that occurred without anthropogenic causes.

DNA Metabarcoding Reveals Diet Overlap between the Endangered Walia Ibex and Domestic Goats - Implications for Conservation.

Human population expansion and associated degradation of the habitat of many wildlife species cause loss of biodiversity and species extinctions. The small Simen Mountains National Park in Ethiopia is one of the last strongholds for the preservation of a number of afro-alpine mammals, plants and birds, and it is home to the rare endemic Walia ibex, Capra walie. The narrow distribution range of this species as well as potential competition for resources with livestock, especially with domestic goat, Capra hircus, may compromise its future survival. Based on a curated afro-alpine taxonomic reference library constructed for plant taxon identification, we investigated the diet of the Walia ibex and addressed the dietary overlap with domestic goat using DNA metabarcoding of faecal samples. Faeces of both species were collected from different localities in the National Park. We show that both species are browsers, with forbs, shrubs and trees comprising the largest proportion of their diet, supplemented by grasses. There was a considerable overlap in dietary preferences. Several of the preferred diet items of the Walia ibex (Alchemilla sp., Hypericum revolutum, Erica arborea and Rumex sp.) were also among the most preferred diet items of the domestic goat. These results indicate that there is potential for competition between the two species, especially during the dry season, when resources are limited. Our findings, in combination with the expected increase in domestic herbivores, suggest that management plans should consider the potential threat posed by domestic goats to ensure future survival of the endangered Walia ibex.

Highly overlapping winter diet in two sympatric lemming species revealed by DNA metabarcoding.

Sympatric species are expected to minimize competition by partitioning resources, especially when these are limited. Herbivores inhabiting the High Arctic in winter are a prime example of a situation where food availability is anticipated to be low, and thus reduced diet overlap is expected. We present here the first assessment of diet overlap of high arctic lemmings during winter based on DNA metabarcoding of feces. In contrast to previous analyses based on microhistology, we found that the diets of both collared (Dicrostonyx groenlandicus) and brown lemmings (Lemmus trimucronatus) on Bylot Island were dominated by Salix while mosses, which were significantly consumed only by the brown lemming, were a relatively minor food item. The most abundant plant taxon, Cassiope tetragona, which alone composes more than 50% of the available plant biomass, was not detected in feces and can thus be considered to be non-food. Most plant taxa that were identified as food items were consumed in proportion to their availability and none were clearly selected for. The resulting high diet overlap, together with a lack of habitat segregation, indicates a high potential for resource competition between the two lemming species. However, Salix is abundant in the winter habitats of lemmings on Bylot Island and the non-Salix portion of the diets differed between the two species. Also, lemming grazing impact on vegetation during winter in the study area is negligible. Hence, it seems likely that the high potential for resource competition predicted between these two species did not translate into actual competition. This illustrates that even in environments with low primary productivity food resources do not necessarily generate strong competition among herbivores.