Dirk Steinke

Barcoding nemo: DNA-based identifications for the ornamental fish trade.

Background Trade in ornamental fishes represents, by far, the largest route for the importation of exotic vertebrates. There is growing pressure to regulate this trade with the goal of ensuring that species are sustainably harvested and that their point of origin is accurately reported. One important element of such regulation involves easy access to specimen identifications, a task that is currently difficult for all but specialists because of the large number of species involved. The present study represents an important first step in making identifications more accessible by assembling a DNA barcode reference sequence library for nearly half of the ornamental fish species imported into North America. Methodology/Principal Findings Analysis of the cytochrome c oxidase subunit I (COI) gene from 391 species from 8 coral reef locations revealed that 98% of these species exhibit distinct barcode clusters, allowing their unambiguous identification. Most species showed little intra-specific variation (adjusted mean = 0.21%), but nine species included two or three lineages showing much more divergence (2.19–6.52%) and likely represent overlooked species complexes. By contrast, three genera contained a species pair or triad that lacked barcode divergence, cases that may reflect hybridization, young taxa or taxonomic over-splitting. Conclusions/Significance Although incomplete, this barcode library already provides a new species identification tool for the ornamental fish industry, opening a realm of applications linked to collection practices, regulatory control and conservation.

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine‐valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA‐based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA‐based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.

Biodiversity of arctic marine fishes: taxonomy and zoogeography

Taxonomic and distributional information on each fish species found in arctic marine waters is reviewed, and a list of families and species with commentary on distributional records is presented. The list incorporates results from examination of museum collections of arctic marine fishes dating back to the 1830s. It also incorporates results from DNA barcoding, used to complement morphological characters in evaluating problematic taxa and to assist in identification of specimens collected in recent expeditions. Barcoding results are depicted in a neighbor-joining tree of 880 CO1 (cytochrome c oxidase 1 gene) sequences distributed among 165 species from the arctic region and adjacent waters, and discussed in the family reviews. Using our definition of the arctic region, we count 242 species with documented presence, if 12 species that likely are synonyms are excluded. The 242 species are distributed among 45 families. Six families in Cottoidei with 72 species and five in Zoarcoidei with 55 species account for more than half (52.5%) the species. This study produced CO1 sequences for 106 of the 242 species. Sequence variability in the barcode region permits discrimination of all species. The average sequence variation within species was 0.3% (range 0–3.5%), while the average genetic distance between congeners was 4.7% (range 3.7–13.3%). The CO1 sequences support taxonomic separation of some species, such as Osmerus dentex and O. mordax and Liparis bathyarcticus and L. gibbus; and synonymy of others, like Myoxocephalus verrucosus in M. scorpius and Gymnelus knipowitschi in G. hemifasciatus. They sometimes revealed the presence of additional species that were not entirely expected, such as an unidentified species of Ammodytes in the western Gulf of Alaska, most likely A. personatus; and an unidentified Icelus species of the I. spatula complex with populations in the western Gulf of Alaska and the northern Bering and Chukchi Seas which could be a new species or a species in synonymy. Reviewing distribution, we found that for 24 species the patterns assigned by authors understated historical presence in the arctic region, and for 12 species they overstated presence. For instance, Hippoglossoides robustus is counted as an arctic–boreal species rather than predominantly boreal, and Artediellus uncinatus as predominantly arctic rather than predominantly boreal. Species with arctic, predominantly arctic, or arctic–boreal distributions composed 41% of the 242 species in the region, and predominantly boreal, boreal, and widely distributed species composed 59%. For some continental shelf species, such as the primarily amphiboreal Eumesogrammus praecisus and Leptoclinus maculatus, distributions appear to reflect changes, including reentry into Arctic seas and reestablishment of continuous ranges, that zoogeographers believe have been going on since the end of land bridge and glacial times.

Taxi - A software tool for DNA barcoding using distance methods

DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding.

Three rounds (1R/2R/3R) of genome duplications and the evolution of the glycolytic pathway in vertebrates

BackgroundEvolution of the deuterostome lineage was accompanied by an increase in systematic complexity especially with regard to highly specialized tissues and organs. Based on the observation of an increased number of paralogous genes in vertebrates compared with invertebrates, two entire genome duplications (2R) were proposed during the early evolution of vertebrates. Most glycolytic enzymes occur as several copies in vertebrate genomes, which are specifically expressed in certain tissues. Therefore, the glycolytic pathway is particularly suitable for testing theories of the involvement of gene/genome duplications in enzyme evolution.ResultsWe assembled datasets from genomic databases of at least nine vertebrate species and at least three outgroups (one deuterostome and two protostomes), and used maximum likelihood and Bayesian methods to construct phylogenies of the 10 enzymes of the glycolytic pathway. Through this approach, we intended to gain insights into the vertebrate specific evolution of enzymes of the glycolytic pathway. Many of the obtained gene trees generally reflect the history of two rounds of duplication during vertebrate evolution, and were in agreement with the hypothesis of an additional duplication event within the lineage of teleost fish. The retention of paralogs differed greatly between genes, and no direct link to the multimeric structure of the active enzyme was found.ConclusionThe glycolytic pathway has subsequently evolved by gene duplication and divergence of each constituent enzyme with taxon-specific individual gene losses or lineage-specific duplications. The tissue-specific expression might have led to an increased retention for some genes since paralogs can subdivide the ancestral expression domain or find new functions, which are not necessarily related to the original function.

Wolbachia and DNA barcoding insects: Patterns, potential, and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein – wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor – for which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.

To be or not to be a flatworm : the acoel controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.

Five years of FISH-BOL: Brief status report

The Fish Barcode of Life Initiative (FISH-BOL) is a concerted global research project launched in 2005, with the goal to collect and assemble standardized DNA barcode sequences and associated voucher provenance data in a curated reference sequence library to aid the molecular identification of all fish species. This article is a detailed progress report (July 2010) on the number of fish species that have been assigned a DNA barcode. Of the approximately 31,000 currently known fish species, 25% have been processed successfully, with at least one species from 89% of all families barcoded; in this report we give a progress overview by taxonomy and geographic region. Using standard analytical protocols, differences in the barcoding completion rate between orders and families are observed, suggesting a potential PCR amplification bias. Overall, between 3 and 9% of the species analyzed failed to yield a “BARCODE compliant” sequence, depending upon how the data are filtered. When species with only a single representative specimen are included, the failure rate was 9%. This might derive from several sources such as mismatched primers and degraded DNA templates. In an attempt to account for the latter, when the analysis is restricted to species with at least two specimens examined, the observed failure rate is significantly lower (3%), suggesting that template quality is a source of concern for FISH-BOL. We, therefore, conclude that using a standard protocol with several specimens per species and PCR primer cocktails is an efficient and successful approach because failures were evenly distributed among orders and families. Only six orders with low species numbers (Pristiformes, Torpediniformes, Albuliformes, Batrachoidiformes, Gobiesociformes, and Petromyzontiformes) showed failure rates between 10 and 33%. Besides outlining an overarching approach for FISH-BOL data curation, the goal of the present article is to give guidance in directing sampling campaigns toward neglected or underrepresented families in order to complete the FISH-BOL campaign most efficiently.

FISH-BOL and seafood identification: Geographically dispersed case studies reveal systemic market substitution across Canada

Background and aims. The Fish Barcode of Life campaign involves a broad international collaboration among scientists working to advance the identification of fishes using DNA barcodes. With over 25% of the worlds known ichthyofauna currently profiled, forensic identification of seafood products is now feasible and is becoming routine. Materials and methods. Driven by growing consumer interest in the food supply, investigative reporters from five different media establishments procured seafood samples (n = 254) from numerous retail establishments located among five Canadian metropolitan areas between 2008 and 2010. The specimens were sent to the Canadian Centre for DNA Barcoding for analysis. By integrating the results from these individual case studies in a summary analysis, we provide a broad perspective on seafood substitution across Canada. Results. Barcodes were recovered from 93% of the samples (n = 236), and identified using the Barcode of Life Data Systems “species identification” engine (www.barcodinglife.org). A 99% sequence similarity threshold was employed as a conservative matching criterion for specimen identification to the species level. Comparing these results against the Canadian Food Inspection Agencys “Fish List” a guideline to interpreting “false, misleading or deceptive” names (as per s 27 of the Fish Inspection regulations) demonstrated that 41% of the samples were mislabeled. Most samples were readily identified; however, this was not true in all cases because some samples had no close match. Others were ambiguous due to limited barcode resolution (or imperfect taxonomy) observed within a few closely related species complexes. The latter cases did not significantly impact the results because even the partial resolution achieved was sufficient to demonstrate mislabeling. Conclusion. This work highlights the functional utility of barcoding for the identification of diverse market samples. It also demonstrates how barcoding serves as a bridge linking scientific nomenclature with approved market names, potentially empowering regulatory bodies to enforce labeling standards. By synchronizing taxonomic effort with sequencing effort and database curation, barcoding provides a molecular identification resource of service to applied forensics.

Comprehensive sampling reveals circumpolarity and sympatry in seven mitochondrial lineages of the Southern Ocean crinoid species Promachocrinus kerguelensis (Echinodermata)

Sampling at appropriate spatial scales in the Southern Ocean is logistically challenging and may influence estimates of diversity by missing intermediate representatives. With the assistance of sampling efforts especially influenced by the International Polar Year 2007–2008, we gathered nearly 1500 specimens of the crinoid species Promachocrinus kerguelensis from around Antarctica. We used phylogeographic and phylogenetic tools to assess its genetic diversity, demographic history and evolutionary relationships. Six phylogroups (A–F) identified in an earlier study are corroborated here, with the addition of one new phylogroup (E2). All phylogroups are circumpolar, sympatric and eurybathic. The phylogeny of Promachocrinus phylogroups reveals two principal clades that may represent two different cryptic species with contrasting demographic histories. Genetic diversity indices vary dramatically within phylogroups, and within populations, suggesting multiple glacial refugia in the Southern Ocean: on the Kerguelen Plateau, in the East Weddell Sea and the South Shetland Islands (Atlantic sector), and on the East Antarctic continental shelf in the Dumont d’Urville Sea and Ross Sea. The inferences of gene flow vary among the phylogroups, showing discordant spatial patterns. Phylogroup A is the only one found in the Sub‐Antarctic region, although without evident connectivity between Bouvet and Kerguelen populations. The Scotia Arc region shows high levels of connectivity between populations in most of the phylogroups, and barriers to gene flow are evident in East Antarctica.