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1.  jMOTU and Taxonerator: Turning DNA Barcode Sequences into Annotated Operational Taxonomic Units 
PLoS ONE  2011;6(4):e19259.
Background
DNA barcoding and other DNA sequence-based techniques for investigating and estimating biodiversity require explicit methods for associating individual sequences with taxa, as it is at the taxon level that biodiversity is assessed. For many projects, the bioinformatic analyses required pose problems for laboratories whose prime expertise is not in bioinformatics. User-friendly tools are required for both clustering sequences into molecular operational taxonomic units (MOTU) and for associating these MOTU with known organismal taxonomies.
Results
Here we present jMOTU, a Java program for the analysis of DNA barcode datasets that uses an explicit, determinate algorithm to define MOTU. We demonstrate its usefulness for both individual specimen-based Sanger sequencing surveys and bulk-environment metagenetic surveys using long-read next-generation sequencing data. jMOTU is driven through a graphical user interface, and can analyse tens of thousands of sequences in a short time on a desktop computer. A companion program, Taxonerator, that adds traditional taxonomic annotation to MOTU, is also presented. Clustering and taxonomic annotation data are stored in a relational database, and are thus amenable to subsequent data mining and web presentation.
Conclusions
jMOTU efficiently and robustly identifies the molecular taxa present in survey datasets, and Taxonerator decorates the MOTU with putative identifications. jMOTU and Taxonerator are freely available from http://www.nematodes.org/.
doi:10.1371/journal.pone.0019259
PMCID: PMC3081837  PMID: 21541350
2.  DNA taxonomy of a neglected animal phylum: an unexpected diversity of tardigrades. 
A molecular survey technique was used to investigate the diversity of terrestrial tardigrades from three sites within Scotland. Ribosomal small subunit sequence was used to classify specimens into molecular operational taxonomic units (MOTU). Most MOTU were identified to the generic level using digital voucher photography. Thirty-two MOTU were defined, a surprising abundance given that the documented British fauna is 68 species. Some tardigrade MOTU were shared between the two rural collection sites, but no MOTU were found in both urban and rural sites, which conflicts with models of ubiquity of meiofaunal taxa. The patterns of relatedness of MOTU were particularly intriguing, with some forming clades with low levels of divergence, suggestive of taxon flocks. Some morphological taxa contained well-separated MOTU, perhaps indicating the existence of cryptic taxa. DNA sequence-based MOTU proved to be a revealing method for meiofaunal diversity studies.
PMCID: PMC1810026  PMID: 15252980
3.  Criconematinae Habitats and Lobocriconema thornei n. sp. (Criconematidae: Nematoda) 
Journal of Nematology  1978;10(1):61-70.
A 16.4-ha arca at the Michigan State University Water Quality Research Site was surveyed to obtain information on the habitats and prominence of taxa of the Criconematinae. Fifteen species representing six genera (Macroposthonia, Lobocriconema, Criconema, Crossonema, Nothocriconema, and Xenocriconemella) of this subfamily were recovered from the experimental site. Species occurrence and population densities were evaluated by using prominence and importance values. The Criconematinae was one of the most prominent and important nematode subfamilies recovered from this area. The species successfully inhabited a broad range of woodlot and field vegetations, and soil management groups. Taxa of the Criconematinae were generally more prominent in woodlot than in field vegetations, although with several important exceptions, especially within the genus Macroposthonia. The second-most prominent and important species recovered was an tandescribed species of Lobocriconerna. It is described as Lobocriconema thornei n. sp., including scanning electron micrographs of females, and descriptions of several of the juvenile stages.
PMCID: PMC2617857  PMID: 19305815
Ecology; Macroposthonia; Lobocriconema; Criconema; Crossonema; Nothocriconema; Xenocriconemella
4.  Revisiting species delimitation within the genus Oxystele using DNA barcoding approach 
ZooKeys  2013;337-354.
The genus Oxystele, a member of the highly diverse marine gastropod superfamily Trochoidea, is endemic to southern Africa. Members of the genus include some of the most abundant molluscs on southern African shores and are important components of littoral biodiversity in rocky intertidal habitats. Species delimitation within the genus is still controversial, especially regarding the complex O. impervia / O. variegata. Here, we assessed species boundaries within the genus using DNA barcoding and phylogenetic tree reconstruction. We analysed 56 specimens using the mitochondrial gene COI. Our analysis delimits five molecular operational taxonomic units (MOTUs), and distinguishes O. impervia from O. variegata. However, we reveal important discrepancies between MOTUs and morphology-based species identification and discuss alternative hypotheses that can account for this. Finally, we indicate the need for future study that includes additional genes, and the combination of both morphology and genetic techniques (e.g. AFLP or microsatellites) to get deeper insight into species delimitation within the genus.
doi:10.3897/zookeys.365.5356
PMCID: PMC3890686  PMID: 24453566
Mollusca; Gastropoda; Trochidae; species delimitation; morphology
5.  DNA barcoding reveals diversity of Hymenoptera and the dominance of parasitoids in a sub-arctic environment 
BMC Ecology  2013;13:2.
Background
Insect diversity typically declines with increasing latitude, but previous studies have shown conflicting latitude-richness gradients for some hymenopteran parasitoids. However, historical estimates of insect diversity and species richness can be difficult to confirm or compare, because they may be based upon dissimilar methods. As a proxy for species identification, we used DNA barcoding to identify molecular operational taxonomic units (MOTUs) for 7870 Hymenoptera specimens collected near Churchill, Manitoba, from 2004 through 2010.
Results
We resolved 1630 MOTUs for this collection, of which 75% (1228) were ichneumonoids (Ichneumonidae + Braconidae) and 91% (1484) were parasitoids. We estimate the total number of Hymenoptera MOTUs in this region at 2624-2840.
Conclusions
The diversity of parasitoids in this sub-Arctic environment implies a high diversity of potential host species throughout the same range. We discuss these results in the contexts of resolving interspecific interactions that may include cryptic species, and developing reproducible methods to estimate and compare species richness across sites and between surveys, especially when morphological specialists are not available to identify every specimen.
doi:10.1186/1472-6785-13-2
PMCID: PMC3565895  PMID: 23351160
Barcoding biotas; Biodiversity; DNA barcoding; Hymenoptera; Sub-Arctic; Parasitoids; Canada
6.  Comparison of Biological, Molecular, and Morphological Methods of Species Identification in a Set of Cultured Panagrolaimus Isolates 
Journal of Nematology  2003;35(1):119-128.
We have developed a molecular barcode system that uses the small subunit ribosomal RNA (SSU) sequence to define molecular operational taxonomic units (MOTU) of soil nematodes. Here we attempt to differentiate five cultured isolates of a taxonomically difficult genus, Panagrolaimus, using morphological, molecular, and biological (breeding) criteria. The results indicated that the five culture populations belonged to two reproductively isolated species. The available morphological criteria, including scanning electron microscopy (SEM), were insufficient to differentiate among them, and all five could be classified as one morphospecies. Within-culture variation of the morphometrical data did not discern between the two biological species. Sequence data clearly separated the populations into two groups that supported the breeding results. Given this study represented only five populations of one genus, we suggest a congruence of MOTU analysis with the biological species concept. This multifaceted approach is promising for future identification of nematodes as it is simple, comparable, and transferable.
PMCID: PMC2620612  PMID: 19265985
biological species concept; breeding; culture isolate; method; molecular barcode; morphology; morphospecies; nematode; Panagrolaimus; rRNA; scanning electron microscopy; taxonomy
7.  Reverse Taxonomy for Elucidating Diversity of Insect-Associated Nematodes: A Case Study with Termites 
PLoS ONE  2012;7(8):e43865.
Background
The molecular operational taxonomic unit (MOTU) has recently been applied to microbial and microscopic animal biodiversity surveys. However, in many cases, some of the MOTUs cannot be definitively tied to any of the taxonomic groups in current databases. To surmount these limitations, the concept of “reverse taxonomy” has been proposed, i.e. to primarily list the MOTUs with morphological information, and then identify and/or describe them at genus/species level using subsamples or by re-isolating the target organisms. Nevertheless, the application of “reverse taxonomy” has not been sufficiently evaluated. Therefore, the practical applicability of “reverse taxonomy” is tested using termite-associated nematodes as a model system for phoretic/parasitic organisms which have high habitat specificity and a potential handle (their termite host species) for re-isolation attempts.
Methodology
Forty-eight species (from 298 colonies) of termites collected from the American tropics and subtropics were examined for their nematode associates using the reverse taxonomy method and culturing attempts (morphological identification and further sequencing efforts). The survey yielded 51 sequence types ( =  MOTUs) belonging to 19 tentatively identified genera. Within these, four were identified based on molecular data with preliminary morphological observation, and an additional seven were identified or characterized from successful culturing, leaving eight genera unidentified.
Conclusions
That 1/3 of the genera were not successfully identified suggests deficiencies in the depth of available sequences in the database and biological characters, i.e. usually isolated as phoretic/parasitic stages which are not available for morphological identification, and too many undiscovered lineages of nematodes. Although there still is the issue of culturability of nematodes, culturing attempts could help to make reverse taxonomy methods more effective. However, expansion of the database, i.e., production of more DNA barcodes tied to biological information by finding and characterizing additional new and known lineages, is necessary for analyzing functional diversity.
doi:10.1371/journal.pone.0043865
PMCID: PMC3429485  PMID: 22952792
8.  DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar 
The role of DNA barcoding as a tool to accelerate the inventory and analysis of diversity for hyperdiverse arthropods is tested using ants in Madagascar. We demonstrate how DNA barcoding helps address the failure of current inventory methods to rapidly respond to pressing biodiversity needs, specifically in the assessment of richness and turnover across landscapes with hyperdiverse taxa. In a comparison of inventories at four localities in northern Madagascar, patterns of richness were not significantly different when richness was determined using morphological taxonomy (morphospecies) or sequence divergence thresholds (Molecular Operational Taxonomic Unit(s); MOTU). However, sequence-based methods tended to yield greater richness and significantly lower indices of similarity than morphological taxonomy. MOTU determined using our molecular technique were a remarkably local phenomenon—indicative of highly restricted dispersal and/or long-term isolation. In cases where molecular and morphological methods differed in their assignment of individuals to categories, the morphological estimate was always more conservative than the molecular estimate. In those cases where morphospecies descriptions collapsed distinct molecular groups, sequence divergences of 16% (on average) were contained within the same morphospecies. Such high divergences highlight taxa for further detailed genetic, morphological, life history, and behavioral studies.
doi:10.1098/rstb.2005.1714
PMCID: PMC1609228  PMID: 16214741
cox1; CO1; Madagascar; collaborative taxonomy; DNA barcode; biodiversity
9.  Criconematina (Nematoda: Tylenchida) from the Aleutian Islands 
Journal of Nematology  1982;14(3):323-331.
A new genus (Cerchnotocriconenta) and three new species (C. psephinum, Hemicycliophora amchitkaensis, and Paratylenchns amundseni) are described from Adak and Amchitka Islands in the Aleutian chain. The new genus differs from all other criconematid genera in having irregular, convex sculpturing consisting of small, oval plates on the anterior and posterior regions of each annule, with the mid-annular region minutely punctate or dentate. H. arnchitkaensis n. sp. resembles H. similis Thorne and H. zuckermani Brzeski, but has only one head annule, instead of two. P. antundseni n. sp., which has a stylet 17-19 μm long, is similar to P. tateae Wu &Townshend and P. labiosus Anderson &Kimpinski, but differs by the presence of males and the possession of conoid-truncate lip region, functional spermatheca, and long male tail (c = 8.5-9.5). Seriespinula seymouri Wu (Mehta &Raski), Nothocrieonema longulum (Gunhold) De Grisse &Loof, and Macroposthonia xenoplax (Raski) De Grisse &Loof are also reported from the islands.
PMCID: PMC2618187  PMID: 19295715
ring nematodes; sheath nematodes; pitt nematodes; taxonomy; ectoparasites
10.  Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda) 
Background
We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms.
Results
DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species.
Conclusion
An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.
doi:10.1186/1742-9994-6-1
PMCID: PMC2657783  PMID: 19128479
11.  DNA Barcodes for Species Identification in the Hyperdiverse Ant Genus Pheidole (Formicidae: Myrmicinae) 
DNA sequencing is increasingly being used to assist in species identification in order to overcome taxonomic impediment. However, few studies attempt to compare the results of these molecular studies with a more traditional species delineation approach based on morphological characters. Mitochondrial DNA Cytochrome oxidase subunit 1 (CO1) gene was sequenced, measuring 636 base pairs, from 47 ants of the genus Pheidole (Formicidae: Myrmicinae) collected in the Brazilian Atlantic Forest to test whether the morphology-based assignment of individuals into species is supported by DNA-based species delimitation. Twenty morphospecies were identified, whereas the barcoding analysis identified 19 Molecular Operational Taxonomic Units (MOTUs). Fifteen out of the 19 DNA-based clusters allocated, using sequence divergence thresholds of 2% and 3%, matched with morphospecies. Both thresholds yielded the same number of MOTUs. Only one MOTU was successfully identified to species level using the CO1 sequences of Pheidole species already in the Genbank. The average pairwise sequence divergence for all 47 sequences was 19%, ranging between 0–25%. In some cases, however, morphology and molecular based methods differed in their assignment of individuals to morphospecies or MOTUs. The occurrence of distinct mitochondrial lineages within morphological species highlights groups for further detailed genetic and morphological studies, and therefore a pluralistic approach using several methods to understand the taxonomy of difficult lineages is advocated.
doi:10.1673/031.013.2701
PMCID: PMC3735075  PMID: 23902257
CO1, DNA-barcoding; morphospecies; MOTU; taxonomy
12.  A Comprehensive DNA Barcode Library for the Looper Moths (Lepidoptera: Geometridae) of British Columbia, Canada 
PLoS ONE  2011;6(3):e18290.
Background
The construction of comprehensive reference libraries is essential to foster the development of DNA barcoding as a tool for monitoring biodiversity and detecting invasive species. The looper moths of British Columbia (BC), Canada present a challenging case for species discrimination via DNA barcoding due to their considerable diversity and limited taxonomic maturity.
Methodology/Principal Findings
By analyzing specimens held in national and regional natural history collections, we assemble barcode records from representatives of 400 species from BC and surrounding provinces, territories and states. Sequence variation in the barcode region unambiguously discriminates over 93% of these 400 geometrid species. However, a final estimate of resolution success awaits detailed taxonomic analysis of 48 species where patterns of barcode variation suggest cases of cryptic species, unrecognized synonymy as well as young species.
Conclusions/Significance
A catalog of these taxa meriting further taxonomic investigation is presented as well as the supplemental information needed to facilitate these investigations.
doi:10.1371/journal.pone.0018290
PMCID: PMC3065486  PMID: 21464900
13.  Distribution Patterns of Some Criconematinae in Different Forest Associations 
Journal of Nematology  1976;8(1):32-35.
A total of 243 samples from Hemlock-Hardwood, Boreal Forest, and Alpine Tundra associations of New York, Vermont, New Hampshire, and Maine were analyzed for species of Bakernema, Criconema, and Criconemoides and for selected edaphic factors. The Hemlock-Hardwood formation contained 13 species of these genera, but the Boreal Forest and Alpine Tundra contained only Criconema menzeli and Criconemoides sphagni. Criconemoides axeste, C. rusticum, and C. xenoplax were associated primarily with mineral soils that have high pH, low moisture after drainage, and organic matter content of less than 15%. Criconemoides sphagni was associated with organic soils that had low pH, high moisture after drainage, and organic matter content greater than 15%.
PMCID: PMC2620147  PMID: 19308192
Boreal Forest; Hemlock-Hardwood; Tundra; pH; organic matter; Bakernema; Criconema; Criconemoides
14.  Barcoding Sponges: An Overview Based on Comprehensive Sampling 
PLoS ONE  2012;7(7):e39345.
Background
Phylum Porifera includes ∼8,500 valid species distributed world-wide in aquatic ecosystems ranging from ephemeral fresh-water bodies to coastal environments and the deep-sea. The taxonomy and systematics of sponges is complicated, and morphological identification can be both time consuming and erroneous due to phenotypic convergence and secondary losses, etc. DNA barcoding can provide sponge biologists with a simple and rapid method for the identification of samples of unknown taxonomic membership. The Sponge Barcoding Project (www.spongebarcoding.org), the first initiative to barcode a non-bilaterian metazoan phylum, aims to provide a comprehensive DNA barcode database for Phylum Porifera.
Methodology/Principal Findings
∼7,400 sponge specimens have been extracted, and amplification of the standard COI barcoding fragment has been attempted for approximately 3,300 museum samples with ∼25% mean amplification success. Based on this comprehensive sampling, we present the first report on the workflow and progress of the sponge barcoding project, and discuss some common pitfalls inherent to the barcoding of sponges.
Conclusion
A DNA-barcoding workflow capable of processing potentially large sponge collections has been developed and is routinely used for the Sponge Barcoding Project with success. Sponge specific problems such as the frequent co-amplification of non-target organisms have been detected and potential solutions are currently under development. The initial success of this innovative project have already demonstrated considerable refinement of sponge systematics, evaluating morphometric character importance, geographic phenotypic variability, and the utility of the standard barcoding fragment for Porifera (despite its conserved evolution within this basal metazoan phylum).
doi:10.1371/journal.pone.0039345
PMCID: PMC3389008  PMID: 22802937
15.  Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding 
By facilitating bioliteracy, DNA barcoding has the potential to improve the way the world relates to wild biodiversity. Here we describe the early stages of the use of cox1 barcoding to supplement and strengthen the taxonomic platform underpinning the inventory of thousands of sympatric species of caterpillars in tropical dry forest, cloud forest and rain forest in northwestern Costa Rica. The results show that barcoding a biologically complex biota unambiguously distinguishes among 97% of more than 1000 species of reared Lepidoptera. Those few species whose barcodes overlap are closely related and not confused with other species. Barcoding also has revealed a substantial number of cryptic species among morphologically defined species, associated sexes, and reinforced identification of species that are difficult to distinguish morphologically. For barcoding to achieve its full potential, (i) ability to rapidly and cheaply barcode older museum specimens is urgent, (ii) museums need to address the opportunity and responsibility for housing large numbers of barcode voucher specimens, (iii) substantial resources need be mustered to support the taxonomic side of the partnership with barcoding, and (iv) hand-held field-friendly barcorder must emerge as a mutualism with the taxasphere and the barcoding initiative, in a manner such that its use generates a resource base for the taxonomic process as well as a tool for the user.
doi:10.1098/rstb.2005.1715
PMCID: PMC1609230  PMID: 16214742
Costa Rica; tropical; Area de Conservación Guanacaste; Hesperiidae; Saturniidae; Sphingidae
16.  DNA barcoding of Northern Nearctic Muscidae (Diptera) reveals high correspondence between morphological and molecular species limits 
BMC Ecology  2012;12:24.
Background
Various methods have been proposed to assign unknown specimens to known species using their DNA barcodes, while others have focused on using genetic divergence thresholds to estimate “species” diversity for a taxon, without a well-developed taxonomy and/or an extensive reference library of DNA barcodes. The major goals of the present work were to: a) conduct the largest species-level barcoding study of the Muscidae to date and characterize the range of genetic divergence values in the northern Nearctic fauna; b) evaluate the correspondence between morphospecies and barcode groupings defined using both clustering-based and threshold-based approaches; and c) use the reference library produced to address taxonomic issues.
Results
Our data set included 1114 individuals and their COI sequences (951 from Churchill, Manitoba), representing 160 morphologically-determined species from 25 genera, covering 89% of the known fauna of Churchill and 23% of the Nearctic fauna. Following an iterative process through which all specimens belonging to taxa with anomalous divergence values and/or monophyly issues were re-examined, identity was modified for 9 taxa, including the reinstatement of Phaonia luteva (Walker) stat. nov. as a species distinct from Phaonia errans (Meigen). In the post-reassessment data set, no distinct gap was found between maximum pairwise intraspecific distances (range 0.00-3.01%) and minimum interspecific distances (range: 0.77-11.33%). Nevertheless, using a clustering-based approach, all individuals within 98% of species grouped with their conspecifics with high (>95%) bootstrap support; in contrast, a maximum species discrimination rate of 90% was obtained at the optimal threshold of 1.2%. DNA barcoding enabled the determination of females from 5 ambiguous species pairs and confirmed that 16 morphospecies were genetically distinct from named taxa. There were morphological differences among all distinct genetic clusters; thus, no cases of cryptic species were detected.
Conclusions
Our findings reveal the great utility of building a well-populated, species-level reference barcode database against which to compare unknowns. When such a library is unavailable, it is still possible to obtain a fairly accurate (within ~10%) rapid assessment of species richness based upon a barcode divergence threshold alone, but this approach is most accurate when the threshold is tuned to a particular taxon.
doi:10.1186/1472-6785-12-24
PMCID: PMC3537539  PMID: 23173946
Insects; Muscid flies; Churchill; Manitoba; Barcoding biotas; Cytochrome c oxidase subunit 1; COI; DNA barcoding; Clustering-based method; Threshold-based method
17.  Integrated Operational Taxonomic Units (IOTUs) in Echolocating Bats: A Bridge between Molecular and Traditional Taxonomy 
PLoS ONE  2012;7(6):e40122.
Background
Nowadays, molecular techniques are widespread tools for the identification of biological entities. However, until very few years ago, their application to taxonomy provoked intense debates between traditional and molecular taxonomists. To prevent every kind of disagreement, it is essential to standardize taxonomic definitions. Along these lines, we introduced the concept of Integrated Operational Taxonomic Unit (IOTU). IOTUs come from the concept of Operational Taxonomic Unit (OTU) and paralleled the Molecular Operational Taxonomic Unit (MOTU). The latter is largely used as a standard in many molecular-based works (even if not always explicitly formalized). However, while MOTUs are assigned solely on molecular variation criteria, IOTUs are identified from patterns of molecular variation that are supported by at least one more taxonomic characteristic.
Methodology/Principal Findings
We tested the use of IOTUs on the widest DNA barcoding dataset of Italian echolocating bats species ever assembled (i.e. 31 species, 209 samples). We identified 31 molecular entities, 26 of which corresponded to the morphologically assigned species, two MOTUs and three IOTUs. Interestingly, we found three IOTUs in Myotis nattereri, one of which is a newly described lineage found only in central and southern Italy. In addition, we found a level of molecular variability within four vespertilionid species deserving further analyses. According to our scheme two of them (i.e. M. bechsteinii and Plecotus auritus) should be ranked as unconfirmed candidate species (UCS).
Conclusions/Significance
From a systematic point of view, IOTUs are more informative than the general concept of OTUs and the more recent MOTUs. According to information content, IOTUs are closer to species, although it is important to underline that IOTUs are not species. Overall, the use of a more precise panel of taxonomic entities increases the clarity in the systematic field and has the potential to fill the gaps between modern and traditional taxonomy.
doi:10.1371/journal.pone.0040122
PMCID: PMC3386196  PMID: 22761951
18.  The promise of a DNA taxonomy. 
Not only is the number of described species a very small proportion of the estimated extant number of taxa, but it also appears that all concepts of the extent and boundaries of 'species' fail in many cases. Using conserved molecular sequences it is possible to define and diagnose molecular operational taxonomic units (MOTU) that have a similar extent to traditional 'species'. Use of a MOTU system not only allows the rapid and effective identification of most taxa, including those not encountered before, but also allows investigation of the evolution of patterns of diversity. A MOTU approach is not without problems, particularly in the area of deciding what level of molecular difference defines a biologically relevant taxon, but has many benefits. Molecular data are extremely well suited to re-analysis and meta-analysis, and data from multiple independent studies can be readily collated and investigated by using new parameters and assumptions. Previous molecular taxonomic efforts have focused narrowly. Advances in high-throughput sequencing methodologies, however, place the idea of a universal, multi-locus molecular barcoding system in the realm of the possible.
PMCID: PMC1693355  PMID: 15253352
19.  Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker 
PLoS ONE  2012;7(8):e42780.
Background
DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.
Methodology/Principal Findings
The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.
Conclusions/Significance
The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.
doi:10.1371/journal.pone.0042780
PMCID: PMC3420951  PMID: 22916158
20.  Revealing the Hyperdiverse Mite Fauna of Subarctic Canada through DNA Barcoding 
PLoS ONE  2012;7(11):e48755.
Although mites are one of the most abundant and diverse groups of arthropods, they are rarely targeted for detailed biodiversity surveys due to taxonomic constraints. We address this gap through DNA barcoding, evaluating acarine diversity at Churchill, Manitoba, a site on the tundra-taiga transition. Barcode analysis of 6279 specimens revealed nearly 900 presumptive species of mites with high species turnover between substrates and between forested and non-forested sites. Accumulation curves have not reached an asymptote for any of the three mite orders investigated, and estimates suggest that more than 1200 species of Acari occur at this locality. The coupling of DNA barcode results with taxonomic assignments revealed that Trombidiformes compose 49% of the fauna, a larger fraction than expected based on prior studies. This investigation demonstrates the efficacy of DNA barcoding in facilitating biodiversity assessments of hyperdiverse taxa.
doi:10.1371/journal.pone.0048755
PMCID: PMC3487733  PMID: 23133656
21.  Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata 
DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover ‘cryptic species’. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be ‘character based’, whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.
doi:10.1098/rspb.2007.1290
PMCID: PMC2212734  PMID: 17999953
character-based DNA barcoding; characteristic attributes organisation system; Odonata; ND1; conservation genetics; biodiversity
22.  Critical factors for assembling a high volume of DNA barcodes 
Large-scale DNA barcoding projects are now moving toward activation while the creation of a comprehensive barcode library for eukaryotes will ultimately require the acquisition of some 100 million barcodes. To satisfy this need, analytical facilities must adopt protocols that can support the rapid, cost-effective assembly of barcodes. In this paper we discuss the prospects for establishing high volume DNA barcoding facilities by evaluating key steps in the analytical chain from specimens to barcodes. Alliances with members of the taxonomic community represent the most effective strategy for provisioning the analytical chain with specimens. The optimal protocols for DNA extraction and subsequent PCR amplification of the barcode region depend strongly on their condition, but production targets of 100K barcode records per year are now feasible for facilities working with compliant specimens. The analysis of museum collections is currently challenging, but PCR cocktails that combine polymerases with repair enzyme(s) promise future success. Barcode analysis is already a cost-effective option for species identification in some situations and this will increasingly be the case as reference libraries are assembled and analytical protocols are simplified.
doi:10.1098/rstb.2005.1727
PMCID: PMC1609220  PMID: 16214753
museum specimens; DNA isolation; PCR; species identification; taxonomy; cox1
23.  A Ranking System for Reference Libraries of DNA Barcodes: Application to Marine Fish Species from Portugal 
PLoS ONE  2012;7(4):e35858.
Background
The increasing availability of reference libraries of DNA barcodes (RLDB) offers the opportunity to the screen the level of consistency in DNA barcode data among libraries, in order to detect possible disagreements generated from taxonomic uncertainty or operational shortcomings. We propose a ranking system to attribute a confidence level to species identifications associated with DNA barcode records from a RLDB. Here we apply the proposed ranking system to a newly generated RLDB for marine fish of Portugal.
Methodology/Principal Findings
Specimens (n = 659) representing 102 marine fish species were collected along the continental shelf of Portugal, morphologically identified and archived in a museum collection. Samples were sequenced at the barcode region of the cytochrome oxidase subunit I gene (COI-5P). Resultant DNA barcodes had average intra-specific and inter-specific Kimura-2-parameter distances (0.32% and 8.84%, respectively) within the range usually observed for marine fishes. All specimens were ranked in five different levels (A–E), according to the reliability of the match between their species identification and the respective diagnostic DNA barcodes. Grades A to E were attributed upon submission of individual specimen sequences to BOLD-IDS and inspection of the clustering pattern in the NJ tree generated. Overall, our study resulted in 73.5% of unambiguous species IDs (grade A), 7.8% taxonomically congruent barcode clusters within our dataset, but awaiting external confirmation (grade B), and 18.7% of species identifications with lower levels of reliability (grades C/E).
Conclusion/Significance
We highlight the importance of implementing a system to rank barcode records in RLDB, in order to flag taxa in need of taxonomic revision, or reduce ambiguities of discordant data. With increasing DNA barcode records publicly available, this cross-validation system would provide a metric of relative accuracy of barcodes, while enabling the continuous revision and annotation required in taxonomic work.
doi:10.1371/journal.pone.0035858
PMCID: PMC3338485  PMID: 22558244
24.  High-throughput sequencing offers insight into mechanisms of resource partitioning in cryptic bat species 
Ecology and Evolution  2011;1(4):556-570.
Sympatric cryptic species, characterized by low morphological differentiation, pose a challenge to understanding the role of interspecific competition in structuring ecological communities. We used traditional (morphological) and novel molecular methods of diet analysis to study the diet of two cryptic bat species that are sympatric in southern England (Plecotus austriacus and P. auritus) (Fig. 1). Using Roche FLX 454 (Roche, Basel, CH) high-throughput sequencing (HTS) and uniquely tagged generic arthropod primers, we identified 142 prey Molecular Operational Taxonomic Units (MOTUs) in the diet of the cryptic bats, 60% of which were assigned to a likely species or genus. The findings from the molecular study supported the results of microscopic analyses in showing that the diets of both species were dominated by lepidopterans. However, HTS provided a sufficiently high resolution of prey identification to determine fine-scale differences in resource use. Although both bat species appeared to have a generalist diet, eared-moths from the family Noctuidae were the main prey consumed. Interspecific niche overlap was greater than expected by chance (Ojk = 0.72, P < 0.001) due to overlap in the consumption of the more common prey species. Yet, habitat associations of nongeneralist prey species found in the diets corresponded to those of their respective bat predator (grasslands for P. austriacus, and woodland for P. auritus). Overlap in common dietary resource use combined with differential specialist prey habitat associations suggests that habitat partitioning is the primary mechanism of coexistence. The performance of HTS is discussed in relation to previous methods of molecular and morphological diet analysis. By enabling species-level identification of dietary components, the application of DNA sequencing to diet analysis allows a more comprehensive comparison of the diet of sympatric cryptic species, and therefore can be an important tool for determining fine-scale mechanisms of coexistence.
doi:10.1002/ece3.49
PMCID: PMC3287336  PMID: 22393522
Diet; interspecific competition; molecular scatology; next generation sequencing; Plecotus
25.  Criconema proclivus n. sp. (Nematoda: Criconematinae) from Woodlands 
Journal of Nematology  1973;5(2):155-157.
Criconema proclivis n. sp. from soil around roots of woodland trees in the northeastern USA is described and illustrated. It is characterized by a total of 67-74 annules, two naked offset head annules, a stylet length of 68.7 -80.7 μ, a sculpted vulval flap, and forward-projecting body annules. The annules at midbody are covered with a continuous fringe of 60-70 spines.
PMCID: PMC2619990  PMID: 19319323
Acer pensylvanicum; Acer saccharum; Betula alleghaniensis; Fagus grandifolia; taxonomy

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