The future of fish-based ecological assessment of European rivers: from traditional EU Water Framework Directive compliant methods to eDNA metabarcoding-based approaches.

Most of the present EU Water Framework Directive (WFD) compliant fish-based assessment methods of European rivers are multi-metric indices computed from traditional electrofishing (TEF) samples, but this method has known shortcomings, especially in large rivers. The probability of detecting rare species remains limited, which can alter the sensitivity of the indices. In recent years, environmental (e)DNA metabarcoding techniques have progressed sufficiently to allow applications in various ecological domains as well as eDNA-based ecological assessment methods. A review of the 25 current WFD-compliant methods for river fish shows that 81% of the metrics used in these methods are expressed in richness or relative abundance and thus compatible with eDNA samples. However, more than half of the member states' methods include at least one metric related to age or size structure and would have to adapt their current fish index if reliant solely on eDNA-derived information. Most trait-based metrics expressed in richness are higher when computed from eDNA than when computed from TEF samples. Comparable values are obtained only when the TEF sampling effort increases. Depending on the species trait considered, most trait-based metrics expressed in relative abundance are significantly higher for eDNA than for TEF samples or vice versa due to over-estimation of sub-surface species or under-estimation of benthic and rare species by TEF sampling, respectively. An existing predictive fish index, adapted to make it compatible with eDNA data, delivers an ecological assessment comparable with the current approved method for 22 of the 25 sites tested. Its associated uncertainty is lower than that of current fish indices. Recommendations for the development of future fish eDNA-based indices and the associated eDNA water sampling strategy are discussed.

Natural Barcodes for Longitudinal Single Cell Tracking of Leukemic and Immune Cell Dynamics.

Blood malignancies provide unique opportunities for longitudinal tracking of disease evolution following therapeutic bottlenecks and for the monitoring of changes in anti-tumor immunity. The expanding development of multi-modal single-cell sequencing technologies affords newer platforms to elucidate the mechanisms underlying these processes at unprecedented resolution. Furthermore, the identification of molecular events that can serve as in-vivo barcodes now facilitate the tracking of the trajectories of malignant and of immune cell populations over time within primary human samples, as these permit unambiguous identification of the clonal lineage of cell populations within heterogeneous phenotypes. Here, we provide an overview of the potential for chromosomal copy number changes, somatic nuclear and mitochondrial DNA mutations, single nucleotide polymorphisms, and T and B cell receptor sequences to serve as personal natural barcodes and review technical implementations in single-cell analysis workflows. Applications of these methodologies include the study of acquired therapeutic resistance and the dissection of donor- and host cellular interactions in the context of allogeneic hematopoietic stem cell transplantation.

Ten years of barcoding at the African Centre for DNA Barcoding.

The African Centre for DNA Barcoding (ACDB) was established in 2005 as part of a global initiative to accurately and rapidly survey biodiversity using short DNA sequences. The mitochondrial cytochrome c oxidase 1 gene (CO1) was rapidly adopted as the de facto barcode for animals. Following the evaluation of several candidate loci for plants, the Plant Working Group of the Consortium for the Barcoding of Life in 2009 recommended that two plastid genes, rbcLa and matK, be adopted as core DNA barcodes for terrestrial plants. To date, numerous studies continue to test the discriminatory power of these markers across various plant lineages. Over the past decade, we at the ACDB have used these core DNA barcodes to generate a barcode library for southern Africa. To date, the ACDB has contributed more than 21 000 plant barcodes and over 3000 CO1 barcodes for animals to the Barcode of Life Database (BOLD). Building upon this effort, we at the ACDB have addressed questions related to community assembly, biogeography, phylogenetic diversification, and invasion biology. Collectively, our work demonstrates the diverse applications of DNA barcoding in ecology, systematics, evolutionary biology, and conservation.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era.

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

What is biodiversity? Stepping forward from barcoding to understanding biological differences.

This opinion paper gives personal views of the direction that cataloguing biodiversity should be going in. Although molecular taxonomy enables rapid and high throughput identification of species, it needs to be anchored to traditional taxonomy, because without information of actual biological properties of species, DNA barcoding just reports differences in selected DNA sequences, which need not have anything to do with the biological properties of the organisms, and the reasons for the development of the species. Since functional differences are the most common reason behind species differences, the future of cataloguing biodiversity and biodiversity research is, in my opinion, in trying to integrate genomic research to comparative physiology in order to be able to evaluate which functional properties have likely been important in generating biodiversity. This task is overwhelming, and requires forgetting the traditional disciplines. Further, major problems associated with the present-day treatment of genomic data are presented from my viewpoint.

Phenotypic vs genotypic approaches to biodiversity, from conflict to alliance.

Taxonomy has traditionally been based on morphological characters. Such a "phenotypic taxonomy" has steadily been replaced by the advent of molecular approaches, culminating with the rapid sequencing of genetic barcodes. The convenience of barcoding and its relative ease has relegated "phenotypic taxonomy" to a historical status. The use of genetics is undeniably powerful. It has relatively few biases and DNA can be extracted from challenging groups, where forms are fragile, such as jellyfish, or where early life stages are difficult to connect with adult forms. The problem is that resources are finite, and the rise of one powerful method came with the demise of traditional taxonomy. In addition, genetic methods may be very sophisticated, requiring acute expertise to master its techniques. These two points in combination have resulted in less funding and attraction for traditional approaches. This is doubly unfortunate because, first we are quickly losing experts in organisms that have incredibly complex lifestyles, and second because in order to fully appreciate a molecular taxonomy, one needs to understand the organisms. In a time of rapid loss of biodiversity, time is ripe for traditional and molecular taxonomists to unite in order to better appreciate and understand the complexity of life forms.

DNA barcoding in Mexico: an introduction.

DNA barcoding has become an important current scientific trend to the understanding of the world biodiversity. In the case of mega-diverse hot spots like Mexico, this technique represents an important tool for taxonomists, allowing them to concentrate in highlighted species by the barcodes instead of analyzing entire sets of specimens. This tendency resulted in the creation of a national network named Mexican Barcode of Life (MEXBOL) which main goals are to train students, and to promote the interaction and collective work among researchers interested in this topic. As a result, the number of records in the Barcode of Life Database (BOLD) for some groups, such as the Mammalia, Actinopterygii, Polychaeta, Branchiopoda, Ostracoda, Maxillopoda, Nematoda, Pinophyta, Ascomycota and Basidiomycota place Mexico among the top ten countries in the generation of these data. This special number presents only few of the many interesting findings in this region of the world, after the use of this technique and its integration with other methodologies.

Future directions.

It is a risky task to attempt to predict the direction that DNA barcoding and its applications may take in the future. In a very short time, the endeavor of DNA barcoding has gone from being a tool to facilitate taxonomy in difficult to identify species, to an ambitious, global initiative that seeks to tackle such pertinent and challenging issues as quantifying global biodiversity, revolutionizing the forensic identifications of species, advancing the study of interactions among species, and promoting the reconstruction of evolutionary relationships within communities. The core element of DNA barcoding will always remain the same: the generation of a set of well-identified samples collected and genotyped at one or more genetic barcode markers and assembled into a properly curated database. But the application of this body of data will depend on the creativity and need of the research community in using a "gold standard" of annotated DNA sequence data at the species level. We foresee several areas where the application of DNA barcode data is likely to yield important evolutionary, ecological, and societal insights, and while far from exclusive, provide examples of how DNA barcode data will continue to empower scientists to address hypothesis-driven research. Three areas of immediate and obvious concern are (1) biodiversity inventories, (2) phylogenetic applications, and (3) species interactions.

An emergent science on the brink of irrelevance: a review of the past 8 years of DNA barcoding.

DNA barcoding has become a well-funded, global enterprise since its proposition as a technique for species identification, delimitation and discovery in 2003. However, the rapid development of next generation sequencing (NGS) has the potential to render DNA barcoding irrelevant because of the speed with which it generates large volumes of genomic data. To avoid obsolescence, the DNA barcoding movement must adapt to use this new technology. This review examines the DNA barcoding enterprise, its continued resistance to improvement and the implications of this on the future of the discipline. We present the consistent failure of DNA barcoding to recognize its limitations and evolve its methodologies, reducing the usefulness of the data produced by the movement and throwing into doubt its ability to embrace NGS.

New insights into molecular evolution: prospects from the Barcode of Life Initiative (BOLI).

Geographic and temporal patterns of morphological and behavioral diversifications among species stimulated Darwin to propose a mechanism for evolutionary change through natural selection. Scientific developments have revealed an even more fundamental level of biological complexity: sequence variation in DNA. While genome projects yield spectacular insights into molecular evolution, they have targeted only a few species. In contrast, the Barcode of Life Initiative (BOLI) proposes a horizontal approach to genomics, examining short, standardized genome segments across the sweep of eukaryotic life, all 10 million species. BOLI will extend our understanding of evolution and speciation in varied ways. It will facilitate quantification of biological diversity by disclosing cryptic species and enabling a rapid survey of taxon diversity in groups that have hitherto received scant morphological examination. It will facilitate assignment of life history stages to known species and provide a first estimate of species ages. It will also reveal key features of the mitochondrial genome, because the evolutionary properties of barcodes relate to those in the mitochondrial genome as a whole, acting to flag taxonomic groups or species with unusual nucleotide composition or evolutionary rates. The growing volume of barcode records has revealed that sequence variability within species is generally much lower than divergence among species (barcoding gap), a pattern that occurs in diverse lineages, suggesting a pervasive evolutionary process. Low variability may reflect recurrent selective sweeps of favored mitochondrial variants propagating as single linkage units across species. If this hypothesis is substantiated, the implications are significant, particularly for our understanding of molecular evolution of mitochondrial DNA and its relationship with species delineation.