The application of DNA sequence data for the identification of benthic nematodes from the North Sea

Nematodes or roundworms represent one of the most diverse and dominant taxon in marine benthic habitats. Whereas a morphological identification of many species is challenging, the application of molecular markers represents a promising approach for species discrimination and identification. In this study, we used an integrative taxonomic approach, combining both molecular and morphological methods, to characterize nematodes of distinct sex and ontogenetic stages from three sampling sites of the North Sea. Morphospecies were discriminated after first visual determination, followed by a molecular analysis of the nuclear 28S rDNA: D2–D3 marker. By linking each sequence to a morphological voucher, discordant morphological identification was subjected to a so-called reverse taxonomic approach. Molecular operational taxonomic units (MOTUs) and morphospecies were compared for all of the three sampling sites to assess concordance of methodology. In total, 32 MOTUs and 26 morphospecies were assigned, of which 12 taxa were identified as described species. Both approaches showed high concordance in taxon assignment (84.4 %) except for a cluster comprising various Sabatieria species. Our study revealed the high potential of the analyzed fragment as a useful molecular marker for the identification of the North Sea nematodes and highlighted the applicability of this combined taxonomic approach in general.     

Assessing Genetic Diversity in Mexican Husk Tomato Species

Mexico is the center of diversity of the husk tomato (Physalis L., Solanaceae), which includes a number of commercially important edible and ornamental species. Taxonomic identification is presently based on morphological characteristics, but the presence of high inter- and intraspecific morphological variation makes this task difficult. Six ISSR primers were used on eight Mexican species of Physalis to determine their utility for interspecific taxonomic discrimination and to assess their potential for inferring interspecific relationships. The six ISSR primers amplified 101 bands, with 100% polymorphism across samples. The number of bands per primer varied from 10 to 21. All primers produced different fingerprint profiles for each species, confirming the ISSR value in taxonomic discrimination. Discrimination values based on Simpson’s diversity index varied from 0.48 to 0.58. Genetic interspecific similarity values ranged from 0.20 to 0.57, and intraspecific similarity values were highest for Physalis angulata (0.71), followed by Physalis philadelphica (0.63) and Physalis lagascae (0.55). The UPGMA analysis grouped accessions of the same species together and clustered together Physalis species of similar morphological traits. Thus, ISSR markers are useful in estimating genetic relationships in Physalis.     

DNA barcoding of African fruit bats (Mammalia, Pteropodidae). The mitochondrial genome does not provide a reliable discrimination between Epomophorus gambianus and Micropteropus pusillus

Sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene have been shown to be useful for species identification in various groups of animals. However, the DNA barcoding approach has never been tested on African fruit bats of the family Pteropodidae (Mammalia, Chiroptera). In this study, the COI gene was sequenced from 120 bats collected in the Central African Republic and belonging to either Epomophorus?gambianus or Micropteropus?pusillus, two species easily diagnosed on the basis of morphological characters, such as body size, skull shape and palatal ridges. Two additional molecular markers were used for comparisons: the complete mitochondrial cytochrome b gene and the intron 7 of the nuclear β-fibrinogen (FGB) gene. Our results reveal an unexpected discordance between mitochondrial and nuclear genes. The nuclear FGB signal agrees with our morphological identifications, as the three alleles detected for E.?gambianus are divergent from the fourteen alleles found for M.?pusillus. By contrast, this taxonomic distinction is not recovered with the analyses of mitochondrial genes, which support rather a polyphyletic pattern for both species. The conflict between molecular markers is explained by multiple mtDNA introgression events from M.?pusillus into E.?gambianus or, alternatively, by incomplete lineage sorting of mtDNA haplotypes associated with positive selection on FGB alleles of M.?pusillus. Our work shows the failure of DNA barcoding to discriminate between two morphologically distinct fruit bat species and highlights the importance of using both mitochondrial and nuclear markers for taxonomic identification.     

Bushmeat genetics: setting up a reference framework for the DNA typing of African forest bushmeat

The bushmeat trade in tropical Africa represents illegal, unsustainable off‐takes of millions of tons of wild game – mostly mammals – per year. We sequenced four mitochondrial gene fragments (cyt b, COI, 12S, 16S) in >300 bushmeat items representing nine mammalian orders and 59 morphological species from five western and central African countries (Guinea, Ghana, Nigeria, Cameroon and Equatorial Guinea). Our objectives were to assess the efficiency of cross‐species PCR amplification and to evaluate the usefulness of our multilocus approach for reliable bushmeat species identification. We provide a straightforward amplification protocol using a single ‘universal’ primer pair per gene that generally yielded >90% PCR success rates across orders and was robust to different types of meat preprocessing and DNA extraction protocols. For taxonomic identification, we set up a decision pipeline combining similarity‐ and tree‐based approaches with an assessment of taxonomic expertise and coverage of the GENBANK database. Our multilocus approach permitted us to: (i) adjust for existing taxonomic gaps in GENBANK databases, (ii) assign to the species level 67% of the morphological species hypotheses and (iii) successfully identify samples with uncertain taxonomic attribution (preprocessed carcasses and cryptic lineages). High levels of genetic polymorphism across genes and taxa, together with the excellent resolution observed among species‐level clusters (neighbour‐joining trees and Klee diagrams) advocate the usefulness of our markers for bushmeat DNA typing. We formalize our DNA typing decision pipeline through an expert‐curated query database – DNAbushmeat – that shall permit the automated identification of African forest bushmeat items.     

Metabarcoding vs. morphological identification to assess diatom diversity in environmental studies

Diatoms are frequently used for water quality assessments; however, identification to species level is difficult, time‐consuming and needs in‐depth knowledge of the organisms under investigation, as nonhomoplastic species‐specific morphological characters are scarce. We here investigate how identification methods based on DNA (metabarcoding using NGS platforms) perform in comparison to morphological diatom identification and propose a workflow to optimize diatom fresh water quality assessments. Diatom diversity at seven different sites along the course of the river system Odra and Lusatian Neisse from the source to the mouth is analysed with DNA and morphological methods, which are compared. The NGS technology almost always leads to a higher number of identified taxa (270 via NGS vs. 103 by light microscopy LM), whose presence could subsequently be verified by LM. The sequence‐based approach allows for a much more graduated insight into the taxonomic diversity of the environmental samples. Taxa retrieval varies considerably throughout the river system, depending on species occurrences and the taxonomic depth of the reference databases. Mostly rare taxa from oligotrophic parts of the river systems are less well represented in the reference database used. A workflow for DNA‐based NGS diatom identification is presented. 28 000 diatom sequences were evaluated. Our findings provide evidence that metabarcoding of diatoms via NGS sequencing of the V4 region (18S) has a great potential for water quality assessments and could complement and maybe even improve the identification via light microscopy.     

Taxonomy of Mechanitis (F.) (Lepidoptera: Nymphalidae) from the West Colombian Andes: an Integrative Approach

Species identification in the butterfly genus Mechanitis (F.) (Lepidoptera: Nymphalidae) becomes difficult when it is based only on wing color patterns, a common practice in butterfly taxonomy. Difficulties in Mechanitis taxonomy are related to the widespread mimicry and polymorphism among species belonging to this genus. Species recognition and inventories of Mechanitis genus in geographic areas as the Andean region of Colombia are of particular interest and the use of more than one character for taxonomic identification is desirable. In this study, we included morphological, ecological, and mitochondrial DNA data to identify the occurring species in this region. Species of Mechanitis were studied from ecological, morphological, and molecular perspectives considering host plant identification, oviposition behavior, and life cycles under laboratory conditions. Immature morphology, patterns of wing color, and genital structures of adults were also studied. The genetic barcoding region of the cytochrome oxidase I mitochondrial gene was sequenced and used to verify the limits between species previously defined by the other characters and to validate its usefulness for species delimitation in this particular genus. The integrative approach combining independent datasets successfully allowed species identification as compared to the approach based on a single dataset. Three well-differentiated species were found in the studied region, Mechanitis menapis (Hewitson), Mechanitis polymnia (Linnaeus), and Mechanitis lysimnia (Fabricius). New valuable characters that could improve taxonomic identification in this genus are considered.     

Molecular identification (RAPD) of the eight species of the genus Barbus (Cyprinidae) in the Iberian Peninsula

Random amplified polymorphic DNA (RAPD) analysis has been used to identify the eight Barbus species of the Iberian Peninsula, whose morphological differentiation is difficult. Ten random primers were employed to generate RAPD markers. Different RAPD profiles were observed for the different species. Four species-specific markers were found in B. bocagei , seven in B. comizo , five in B. graellsii , three in B. guiraonis , eight in B. haasi , 13 in B. meridionalis , four in B. microcephalus and four in B. sclateri. Evidence is presented that RAPD markers constitute useful tools for accurate taxonomic identification of Spanish barbels which is one of the first prerequisites in effective conservation programmes.     

Increased accuracy of species lists developed for alpine lakes using morphology and cytochrome oxidase I for identification of specimens

The first step in many community ecology studies is to produce a species list from a sample of individuals. Community ecologists now have two viable ways of producing a species list: morphological and barcode identification. In this study, we compared the taxonomic resolution gained by a combined use of both methods and tested whether a change in taxonomic resolution significantly impacted richness estimates for benthic macroinvertebrates sampled from ten lakes in Sequoia National Park, USA. Across all lakes, 77 unique taxa were identified and 42% (32) were reliably identified to species using both barcode and morphological identification. Of the 32 identified to species, 63% (20) were identified solely by comparing the barcode sequence from cytochrome oxidase I to the Barcode of Life reference library. The increased resolution using a combined identification approach compared to identifications based solely on morphology resulted in a significant increase in estimated richness within a lake at the order, family, genus and species levels of taxonomy (P < 0.05). Additionally, young or damaged individuals that could not be identified using morphology were identified using their COI sequences to the genus or species level on average 75% of the time. Our results demonstrate that a combined identification approach improves accuracy of benthic macroinvertebrate species lists in alpine lakes and subsequent estimates of richness. We encourage the use of barcodes for identification purposes and specifically when morphology is insufficient, as in the case of damaged and early life stage specimens of benthic macroinvertebrates.     

High-Throughput Sequencing—The Key to Rapid Biodiversity Assessment of Marine Metazoa?

The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of the V1-V2 region was used to analyze two pooled samples of DNA, one comprising 118 and the other 37 morphologically identified species, and one natural sample taken directly from a North Sea zooplankton community. A DNA reference library comprising all species represented in the pooled samples was created by Sanger sequencing, and this was then used to determine the optimal similarity threshold for species delineation. The optimal threshold was found at 99% species similarity, with 85% identification success. Pyrosequencing was able to identify between fewer species: 67% and 78% of the species in the two pooled samples. Also, a large number of sequences for three species that were not included in the pooled samples were amplified by pyrosequencing, suggesting preferential amplification of some genotypes and the sensitivity of this approach to even low levels of contamination. Conversely, metagenetic analysis of the natural zooplankton sample identified many more species (particularly gelatinous zooplankton and meroplankton) than morphological analysis of a formalin-fixed sample from the same sampling site, suggesting an increased level of taxonomic resolution with pyrosequencing. The study demonstrated that, based on the V1-V2 region, 454 sequencing does not provide accurate species differentiation and reliable taxonomic classification, as it is required in most biodiversity monitoring. The analysis of artificially prepared samples indicated that species detection in pyrosequencing datasets is complicated by potential PCR-based biases and that the V1-V2 marker is poorly resolved for some taxa.     

Discrimination of Cricotopus species (Diptera: Chironomidae) by DNA barcoding

Chironomids (Diptera) typically comprise the most abundant group of macroinvertebrates collected in water quality surveys. Species in the genus Cricotopus display a wide range of tolerance for manmade pollutants, making them excellent bioindicators. Unfortunately, the usefulness of Cricotopus is overshadowed by the difficulty of accurately identifying larvae using current morphological keys. Molecular approaches are now being used for identification and taxonomic resolution in many animal taxa. In this study, a sequence-based approach for the mitochondrial gene, cytochrome oxidase I (COI), was developed to facilitate identification of Cricotopus species collected from Baltimore area streams. Using unique COI sequence variations, we developed profiles for seven described Cricotopus sp., four described Orthocladius sp., one described Paratrichocladius sp. and one putative species of Cricotopus. In addition to providing an accurate method for identification of Cricotopus, this method will make a useful contribution to the development of keys for Nearctic Cricotopus.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

An integrative approach to species discovery in odonates: from character‐based DNA barcoding to ecology

Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision‐making. Such an approach can enhance both species identification and species discovery. The character‐based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA‐barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.     

Novel DNA markers reveal for taxonomic characterization and identification of the Fusarium fungi species

RAPD analysis for ten F. sporotrichioides strains of different geographical origin was done for DNA loci, potentially suitable as a new markers for taxonomic characterization and identification of toxigenic Fusarium fungi. Three selected monomorphic fragments--products of amplification with one of standard RAPD primers were sequenced that allowed creating SCAR markers for identification of Fusarium fungi on the species group level with similar profiles of produced mycotoxins.     

Comparing Molecular Variation to Morphological Species Designations in the Deep-Sea Coral Narella Reveals New Insights into Seamount Coral Ranges

Recent studies have countered the paradigm of seamount isolation, confounding conservation efforts at a critical time. Efforts to study deep-sea corals, one of the dominant taxa on seamounts, to understand seamount connectivity, are hampered by a lack of taxonomic keys. A prerequisite for connectivity is species overlap. Attempts to better understand species overlap using DNA barcoding methods suggest coral species are widely distributed on seamounts and nearby features. However, no baseline has been established for variation in these genetic markers relative to morphological species designations for deep-sea octocoral families. Here we assess levels of genetic variation in potential octocoral mitochondrial barcode markers relative to thoroughly examined morphological species in the genus Narella. The combination of six markers used here, approximately 3350 bp of the mitochondrial genome, resolved 83% of the morphological species. Our results show that two of the markers, ND2 and NCR1, are not sufficient to resolve genera within Primnoidae, let alone species. Re-evaluation of previous studies of seamount octocorals based on these results suggest that those studies were looking at distributions at a level higher than species, possibly even genus or subfamily. Results for Narella show that using more markers provides haplotypes with relatively narrow depth ranges on the seamounts studied. Given the lack of 100% resolution of species with such a large portion of the mitochondrial genome, we argue that previous genetic studies have not resolved the degree of species overlap on seamounts and that we may not have the power to even test the hypothesis of seamount isolation using mitochondrial markers, let alone refute it. Thus a precautionary approach is advocated in seamount conservation and management, and the potential for depth structuring should be considered.     

A preliminary RAPD-PCR analysis of Cimicifuga species and other botanicals used for women''s health.

Traditional taxonomic methods of botanical identification that rely primarily on morphological observations cannot be used efficiently when only powdered plant materials are available. Thus, our objectives were to determine if we could apply a molecular approach to: a) produce unique DNA profiles that are characteristic of the species, and b) determine if the geographical area or time of collection influences these DNA profiles. Towards this end, random amplified polymorphic DNA (RAPD) analyses were performed on a number of botanicals currently used for women's health. The test materials included samples from three species each of the genera Cimicifuga (Actaea) and Trifolium, as well as samples of Vitex agnus-castus L., Glycyrrhiza glabra L., Gingko biloba L., Valeriana officinalis L., Angelica sinensis (Oliv.) Diels, Viburnum prunifolium L., Humulus lupulus L., Vaccinium macrocarpon Ait., Panax ginseng C.A. Mey. Cimicifuga racemosa (L.) Nutt. and Trifolium pratense L. are currently under clinical investigation in our basic research laboratories and medical clinic for the relief of post-menopausal symptoms. Characteristic profiles produced with the OPC-15 primer could distinguish the three Cimicifuga species: C. racemosa, C. americana and C. rubifolia. Similar results were obtained with the three Trifolium species: Trifolium pratense L., Trifolium incarnatum L., and Trifolium repens L. Accessions of cultivated T. pratense collected from the same field at different times, produced identical profiles. Accessions of Cimicifuga species collected from different geographical areas produced similar but not identical DNA profiles; however, species-specific DNA fragments were identified. These results demonstrate that RAPD analysis can be applied to distinguish species when only powdered material is available for testing. This methodology can be applied to identify species of commercial value regardless of collection time or geographic area.     

Some ‘ant’swers: Application of a layered barcode approach to problems in ant taxonomy

DNA barcoding has emerged as a routine tool in modern taxonomy. Although straightforward, this approach faces new challenges, when applied to difficult situation such as defining cryptic biodiversity. Ants are prime examples for high degrees of cryptic biodiversity due to complex population differentiation, hybridization and speciation processes. Here, we test the DNA barcoding region, cytochrome c oxidase 1 and two supplementary markers, 28S ribosomal DNA and long‐wavelength rhodopsin, commonly used in ant taxonomy, for their potential in a layered, character‐based barcoding approach across different taxonomic levels. Furthermore, we assess performance of the character‐based barcoding approach to determine cryptic species diversity in ants. We found (i) that the barcode potential of a specific genetic marker varied widely among taxonomic levels in ants; (ii) that application of a layered, character‐based barcode for identification of specimens can be a solution to taxonomical challenging groups; (iii) that the character‐based barcoding approach allows us to differentiate specimens even within locations based on pure characters. In summary, (layered) character‐based barcoding offers a reliable alternative for problematic species identification in ants and can be used as a fast and cost‐efficient approach to estimate presence, absence or frequency of cryptic species.     

Comparison of taxonomic, colony morphotype and PCR-RFLP methods to characterize microfungal diversity

We compared three methods for estimating fungal species diversity in soil samples. A rapid screening method based on gross colony morphological features and color reference standards was compared with traditional fungal taxonomic methods and PCR-RFLP for estimation of ecological indices of soil microfungal community composition. Normalized counts of colony morphotypes on dichloran rose bengal medium were used to estimate species richness (S) and evenness (J) and to calculate Shannon's diversity (H) and Simpson's (SI) dominance indices. Isolates were obtained by dilution plating techniques from litter and soil layer samples taken from Douglas-fir forest and clear-cut areas at two locations in the Cascade Mountains. The highest correspondence (97%) was observed between taxonomic identification and RFLP patterns (32:33). Cladistic analyses of PCR-RFLP patterns indicated an 81% correspondence between RFLP patterns:colony morphotypes (33:41). A correspondence of 78% was observed between traditional taxonomic identification:colony morphotypes (32:41). Statistical analyses of ecological indices based on quantitative application of the colony morphotyping method indicated significant differences (P < 0.05) in fungal community composition between forested and clear-cut areas at the Toad Road site but not at the Falls Creek site. Comparisons of ecological indices based on traditional identification of taxa by microscopic characterization on defined culture media resulted in identical findings of statistical significance. The colony morphotyping approach is proposed as a screening method to identify potential effects of land management practices, edaphic factors and pollutants on microfungal diversity.     

The rapid identification of Acinetobacter species using Fourier transform infrared spectroscopy

AIMS: Fourier transform infrared (FT-IR) was used to analyse a selection of Acinetobacter isolates in order to determine if this approach could discriminate readily between the known genomic species of this genus and environmental isolates from activated sludge. METHODS AND RESULTS: FT-IR spectroscopy is a rapid whole-organism fingerprinting method, typically taking only 10 s per sample, and generates 'holistic' biochemical profiles (or 'fingerprints') from biological materials. The cluster analysis produced by FT-IR was compared with previous polyphasic taxonomic studies on these isolates and with 16S-23S rDNA intergenic spacer region (ISR) fingerprinting presented in this paper. FT-IR and 16S-23S rDNA ISR analyses together indicate that some of the Acinetobacter genomic species are particularly heterogeneous and poorly defined, making characterization of the unknown environmental isolates with the genomic species difficult. CONCLUSIONS: Whilst the characterization of the isolates from activated sludge revealed by FT-IR and 16S-23S rDNA ISR were not directly comparable, the dendrogram produced from FT-IR data did correlate well with the outcomes of the other polyphasic taxonomic work. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe it would be advantageous to pursue this approach further and establish a comprehensive database of taxonomically well-defined Acinetobacter species to aid the identification of unknown strains. In this instance, FT-IR may provide the rapid identification method eagerly sought for the routine identification of Acinetobacter isolates from a wide range of environmental sources.     

A continent of plant defense peptide diversity: cyclotides in Australian Hybanthus (Violaceae)

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.