GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates.

Here we describe GoFish, a strategy for single-species environmental DNA (eDNA) presence/absence assays using nested PCR. The assays amplify a mitochondrial 12S rDNA segment with vertebrate metabarcoding primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens for 36 northwestern Atlantic taxa not well documented in GenBank. Using these and existing 12S records, we designed GoFish assays for 11 bony fish species common in the lower Hudson River estuary and tested seasonal abundance and habitat preference at two sites. Additional assays detected nine cartilaginous fish species and a marine mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. From water collection to Sanger sequencing results, the assay can be carried out in three days. The main limitations to this approach, which employs metabarcoding primers, are the same as for metabarcoding, namely, inability to distinguish species with shared target sequences and inconsistent amplification of rarer eDNA. In addition, the performance of the 20 assays reported here as compared to other single-species eDNA assays is not known. This approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings.

Aquatic environmental DNA detects seasonal fish abundance and habitat preference in an urban estuary.

The difficulty of censusing marine animal populations hampers effective ocean management. Analyzing water for DNA traces shed by organisms may aid assessment. Here we tested aquatic environmental DNA (eDNA) as an indicator of fish presence in the lower Hudson River estuary. A checklist of local marine fish and their relative abundance was prepared by compiling 12 traditional surveys conducted between 1988-2015. To improve eDNA identification success, 31 specimens representing 18 marine fish species were sequenced for two mitochondrial gene regions, boosting coverage of the 12S eDNA target sequence to 80% of local taxa. We collected 76 one-liter shoreline surface water samples at two contrasting estuary locations over six months beginning in January 2016. eDNA was amplified with vertebrate-specific 12S primers. Bioinformatic analysis of amplified DNA, using a reference library of GenBank and our newly generated 12S sequences, detected most (81%) locally abundant or common species and relatively few (23%) uncommon taxa, and corresponded to seasonal presence and habitat preference as determined by traditional surveys. Approximately 2% of fish reads were commonly consumed species that are rare or absent in local waters, consistent with wastewater input. Freshwater species were rarely detected despite Hudson River inflow. These results support further exploration and suggest eDNA will facilitate fine-scale geographic and temporal mapping of marine fish populations at relatively low cost.

Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies.

DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648-bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein-encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most - possibly all - synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well-curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.

Interbreeding among deeply divergent mitochondrial lineages in the American cockroach (Periplaneta americana).

DNA barcoding promises to be a useful tool to identify pest species assuming adequate representation of genetic variants in a reference library. Here we examined mitochondrial DNA barcodes in a global urban pest, the American cockroach (Periplaneta americana). Our sampling effort generated 284 cockroach specimens, most from New York City, plus 15 additional U.S. states and six other countries, enabling the first large-scale survey of P. americana barcode variation. Periplaneta americana barcode sequences (n = 247, including 24 GenBank records) formed a monophyletic lineage separate from other Periplaneta species. We found three distinct P. americana haplogroups with relatively small differences within (≤0.6%) and larger differences among groups (2.4%-4.7%). This could be interpreted as indicative of multiple cryptic species. However, nuclear DNA sequences (n = 77 specimens) revealed extensive gene flow among mitochondrial haplogroups, confirming a single species. This unusual genetic pattern likely reflects multiple introductions from genetically divergent source populations, followed by interbreeding in the invasive range. Our findings highlight the need for comprehensive reference databases in DNA barcoding studies, especially when dealing with invasive populations that might be derived from multiple genetically distinct source populations.

DNA barcoding works in practice but not in (neutral) theory.

BACKGROUND: DNA barcode differences within animal species are usually much less than differences among species, making it generally straightforward to match unknowns to a reference library. Here we aim to better understand the evolutionary mechanisms underlying this usual "barcode gap" pattern. We employ avian barcode libraries to test a central prediction of neutral theory, namely, intraspecific variation equals 2 Nµ, where N is population size and µ is mutations per site per generation. Birds are uniquely suited for this task: they have the best-known species limits, are well represented in barcode libraries, and, most critically, are the only large group with documented census population sizes. In addition, we ask if mitochondrial molecular clock measurements conform to neutral theory prediction of clock rate equals µ. RESULTS: Intraspecific COI barcode variation was uniformly low regardless of census population size (n = 142 species in 15 families). Apparent outliers reflected lumping of reproductively isolated populations or hybrid lineages. Re-analysis of a published survey of cytochrome b variation in diverse birds (n = 93 species in 39 families) further confirmed uniformly low intraspecific variation. Hybridization/gene flow among species/populations was the main limitation to DNA barcode identification. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first large study of animal mitochondrial diversity using actual census population sizes and the first to test outliers for population structure. Our finding of universally low intraspecific variation contradicts a central prediction of neutral theory and is not readily accounted for by commonly proposed ad hoc modifications. We argue that the weight of evidence-low intraspecific variation and the molecular clock-indicates neutral evolution plays a minor role in mitochondrial sequence evolution. As an alternate paradigm consistent with empirical data, we propose extreme purifying selection, including at synonymous sites, limits variation within species and continuous adaptive selection drives the molecular clock.

TreeParser-aided Klee diagrams display taxonomic clusters in DNA barcode and nuclear gene datasets.

Indicator vector analysis of a nucleotide sequence alignment generates a compact heat map, called a Klee diagram, with potential insight into clustering patterns in evolution. However, so far this approach has examined only mitochondrial cytochrome c oxidase I (COI) DNA barcode sequences. To further explore, we developed TreeParser, a freely-available web-based program that sorts a sequence alignment according to a phylogenetic tree generated from the dataset. We applied TreeParser to nuclear gene and COI barcode alignments from birds and butterflies. Distinct blocks in the resulting Klee diagrams corresponded to species and higher-level taxonomic divisions in both groups, and this enabled graphic comparison of phylogenetic information in nuclear and mitochondrial genes. Our results demonstrate TreeParser-aided Klee diagrams objectively display taxonomic clusters in nucleotide sequence alignments. This approach may help establish taxonomy in poorly studied groups and investigate higher-level clustering which appears widespread but not well understood.

Frequency matrix approach demonstrates high sequence quality in avian BARCODEs and highlights cryptic pseudogenes.

The accuracy of DNA barcode databases is critical for research and practical applications. Here we apply a frequency matrix to assess sequencing errors in a very large set of avian BARCODEs. Using 11,000 sequences from 2,700 bird species, we show most avian cytochrome c oxidase I (COI) nucleotide and amino acid sequences vary within a narrow range. Except for third codon positions, nearly all (96%) sites were highly conserved or limited to two nucleotides or two amino acids. A large number of positions had very low frequency variants present in single individuals of a species; these were strongly concentrated at the ends of the barcode segment, consistent with sequencing error. In addition, a small fraction (0.1%) of BARCODEs had multiple very low frequency variants shared among individuals of a species; these were found to represent overlooked cryptic pseudogenes lacking stop codons. The calculated upper limit of sequencing error was 8 × 10(-5) errors/nucleotide, which was relatively high for direct Sanger sequencing of amplified DNA, but unlikely to compromise species identification. Our results confirm the high quality of the avian BARCODE database and demonstrate significant quality improvement in avian COI records deposited in GenBank over the past decade. This approach has potential application for genetic database quality control, discovery of cryptic pseudogenes, and studies of low-level genetic variation.

DNA barcoding birds: from field collection to data analysis.

As of February 2011, COI DNA barcode sequences (a 648-bp segment of the 5' end of the mitochondrial gene cytochrome c oxidase I, the standard DNA barcode for animals) have been collected from over 23,000 avian specimens representing 3,800 species, more than one-third of the world's avifauna. Here, we detail the methodology for obtaining DNA barcodes from birds, covering the entire process from field collection to data analysis. We emphasize key aspects of the process and describe in more detail those that are particularly relevant in the case of birds. We provide elemental information about collection of specimens, detailed protocols for DNA extraction and PCR, and basic aspects of sequencing methodology. In particular, we highlight the primer pairs and thermal cycling profiles associated with successful amplification and sequencing from a broad range of avian species. Finally, we succinctly review the methodology for data analysis, including the detection of errors (such as contamination, misidentifications, or amplification of pseudogenes), assessment of species resolution, detection of divergent intraspecific lineages, and identification of unknown specimens.

Commercial teas highlight plant DNA barcode identification successes and obstacles.

Appearance does not easily identify the dried plant fragments used to prepare teas to species. Here we test recovery of standard DNA barcodes for land plants from a large array of commercial tea products and analyze their performance in identifying tea constituents using existing databases. Most (90%) of 146 tea products yielded rbcL or matK barcodes using a standard protocol. Matching DNA identifications to listed ingredients was limited by incomplete databases for the two markers, shared or nearly identical barcodes among some species, and lack of standard common names for plant species. About 1/3 of herbal teas generated DNA identifications not found on labels. Broad scale adoption of plant DNA barcoding may require algorithms that place search results in context of standard plant names and character-based keys for distinguishing closely-related species. Demonstrating the importance of accessible plant barcoding, our findings indicate unlisted ingredients are common in herbal teas.

Project description: DNA barcodes of bird species in the national museum of natural history, smithsonian institution, USA.

The Division of Birds, National Museum of Natural History, Smithsonian Institution in Washington, DC, has obtained and released DNA barcodes for 2808 frozen tissue samples. Of the 1,403 species represented by these samples, 1,147 species have not been barcoded previously. This data release increases the number of bird species with standard barcodes by 91%. These records meet the data standard of the Consortium for the Barcode of Life and they have the reserved keyword BARCODE in GenBank. The data are now available on GenBank and the Barcode of Life Data Systems.

Molecular species identification of Central European ground beetles (Coleoptera: Carabidae) using nuclear rDNA expansion segments and DNA barcodes.

BACKGROUND: The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI) gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous. RESULTS: We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97%) of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95%) of the studied Carabidae. CONCLUSION: Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.

Structural analysis of biodiversity.

Large, recently-available genomic databases cover a wide range of life forms, suggesting opportunity for insights into genetic structure of biodiversity. In this study we refine our recently-described technique using indicator vectors to analyze and visualize nucleotide sequences. The indicator vector approach generates correlation matrices, dubbed Klee diagrams, which represent a novel way of assembling and viewing large genomic datasets. To explore its potential utility, here we apply the improved algorithm to a collection of almost 17,000 DNA barcode sequences covering 12 widely-separated animal taxa, demonstrating that indicator vectors for classification gave correct assignment in all 11,000 test cases. Indicator vector analysis revealed discontinuities corresponding to species- and higher-level taxonomic divisions, suggesting an efficient approach to classification of organisms from poorly-studied groups. As compared to standard distance metrics, indicator vectors preserve diagnostic character probabilities, enable automated classification of test sequences, and generate high-information density single-page displays. These results support application of indicator vectors for comparative analysis of large nucleotide data sets and raise prospect of gaining insight into broad-scale patterns in the genetic structure of biodiversity.

A scalable method for analysis and display of DNA sequences.

BACKGROUND: Comparative DNA sequence analysis provides insight into evolution and helps construct a natural classification reflecting the Tree of Life. The growing numbers of organisms represented in DNA databases challenge tree-building techniques and the vertical hierarchical classification may obscure relationships among some groups. Approaches that can incorporate sequence data from large numbers of taxa and enable visualization of affinities across groups are desirable. METHODOLOGY/PRINCIPAL FINDINGS: Toward this end, we developed a procedure for extracting diagnostic patterns in the form of indicator vectors from DNA sequences of taxonomic groups. In the present instance the indicator vectors were derived from mitochondrial cytochrome c oxidase I (COI) sequences of those groups and further analyzed on this basis. In the first example, indicator vectors for birds, fish, and butterflies were constructed from a training set of COI sequences, then correlations with test sequences not used to construct the indicator vector were determined. In all cases, correlation with the indicator vector correctly assigned test sequences to their proper group. In the second example, this approach was explored at the species level within the bird grouping; this also gave correct assignment, suggesting the possibility of automated procedures for classification at various taxonomic levels. A false-color matrix of vector correlations displayed affinities among species consistent with higher-order taxonomy. CONCLUSIONS/SIGNIFICANCE: The indicator vectors preserved DNA character information and provided quantitative measures of correlations among taxonomic groups. This method is scalable to the largest datasets envisioned in this field, provides a visually-intuitive display that captures relational affinities derived from sequence data across a diversity of life forms, and is potentially a useful complement to current tree-building techniques for studying evolutionary processes based on DNA sequence data.

Comprehensive DNA barcode coverage of North American birds.

DNA barcoding seeks to assemble a standardized reference library for DNA-based identification of eukaryotic species. The utility and limitations of this approach need to be tested on well-characterized taxonomic assemblages. Here we provide a comprehensive DNA barcode analysis for North American birds including 643 species representing 93% of the breeding and pelagic avifauna of the USA and Canada. Most (94%) species possess distinct barcode clusters, with average neighbour-joining bootstrap support of 98%. In the remaining 6%, barcode clusters correspond to small sets of closely related species, most of which hybridize regularly. Fifteen (2%) currently recognized species are comprised of two distinct barcode clusters, many of which may represent cryptic species. Intraspecific variation is weakly related to census population size and species age. This study confirms that DNA barcoding can be effectively applied across the geographical and taxonomic expanse of North American birds. The consistent finding of constrained intraspecific mitochondrial variation in this large assemblage of species supports the emerging view that selective sweeps limit mitochondrial diversity.

Identification of Birds through DNA Barcodes.

Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10x average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.