Barcoding Studies Research Articles

Species delimitation and evolutionary relationships among Phoebis New World sulphur butterflies (Lepidoptera, Pieridae, Coliadinae)

ABSTRACTThe most accepted taxonomic treatment of the New World sulphurs of the genus Phoebis Hübner, [1819] recognizes 16 species including those in the current synonyms Aphrissa and Rhabdodryas. This total conflicts with the results of several recent pierid DNA barcode studies across the Neotropics. We used a five‐locus dataset to carry out species delimitation analyses using the coalescence‐based method implemented in bpp software. After testing the resulting species hypotheses using marginal likelihood estimates, we inferred their phylogenetic relationships and performed an ancestral range reconstruction with biogeobears. Our analyses recovered two different hypotheses, 26 and 24 species, that scored the highest marginal likelihood estimate. Differences between these two hypotheses, when reconciled with barcode clusters and morphology, indicated that 24 is the most likely number of species. Phoebis neocypris stat. rev., Phoebis rurina stat. rev., Phoebis virgo stat. rev., Phoebis marcellina stat. rev., Phoebis thalestris stat. rev., and Phoebis rorata stat. rev. are raised to the species rank. We dated the crown age of Phoebis to the mid‐Miocene, with the islands of the Greater Antilles as the most probable ancestral range. Three main clades of Phoebis diverged early in the evolutionary history of the genus, but most extant species‐level diversity arose after the Pliocene–Pleistocene boundary. Our analyses recovered alternate range expansions and contractions, and dispersal from the islands to the continent and back, in the three main clades. Both sympatric and allopatric speciation seem to have shaped the current species richness.

Chloroplast Genome-Based Hypervariable Markers for Rapid Authentication of Six Korean Pyropia Species

We previously established that polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using partial plastid rbcL and mitochondrial trnC–trnP gene sequences can be used to distinguish the six representative Pyropia species produced via mariculture in Korea. In this study, we develop progressive InDel markers by comparing seven complete Pyropia chloroplast genomes obtained from The National Center of Biotechnology Informnation (NCBI) GenBank. Comparative analyses of nucleotide diversity among the genomes revealed seven hypervariable sites (cemA, rps13, trnM-argB, petD-petB, trnR-trnQ, ccs1-orf24, and ycf12-ftrB) among 637 sliding windows with nucleotide diversity > 0.025 (Pi). These sites included two genes and five gene-intergenic regions, three of which (cemA, trnM-argB, trnR-trnQ) showed complete amplification for all six test species. Finally, trnM-argB, an InDel-variable locus with high discriminatory power, was selected as a DNA barcode candidate. These results suggest that the obtained trnM-argB region can be used for the effective exploration of the variation present in six Korean Pyropia and for further evolutionary, phylogenetic, barcoding and genetic engineering studies of Pyropia species.

Broad North Atlantic distribution of a meiobenthic annelid – against all odds

DNA barcoding and population genetic studies have revealed an unforeseen hidden diversity of cryptic species among microscopic marine benthos, otherwise exhibiting highly similar and simple morphologies. This has led to a paradigm shift, rejecting cosmopolitism of marine meiofauna until genetically proven and challenging the “Everything is Everywhere, but the environment selects” hypothesis that claims ubiquitous distribution of microscopic organisms. With phylogenetic and species delimitation analyses of worldwide genetic samples of the meiofaunal family Dinophilidae (Annelida) we here resolve three genera within the family and showcase an exceptionally broad, boreal, North Atlantic distribution of a single microscopic marine species with no obvious means of dispersal besides vicariance. With its endobenthic lifestyle, small size, limited migratory powers and lack of pelagic larvae, the broad distribution of Dinophilus vorticoides seems to constitute a “meiofaunal paradox”. This species feasts in the biofilm among sand grains, but also on macroalgae and ice within which it can likely survive long-distance rafting dispersal due to its varying lifecycle stages; eggs encapsulated in cocoons and dormant encystment stages. Though often neglected and possibly underestimated among marine microscopic species, dormancy may be a highly significant factor for explaining wide distribution patterns and a key to solving this meiofaunal paradox.

Automated, phylogeny-based genotype delimitation of the Hepatitis Viruses HBV and HCV.

BackgroundThe classification of hepatitis viruses still predominantly relies on ad hoc criteria, i.e., phenotypic traits and arbitrary genetic distance thresholds. Given the subjectivity of such practices coupled with the constant sequencing of samples and discovery of new strains, this manual approach to virus classification becomes cumbersome and impossible to generalize.MethodsUsing two well-studied hepatitis virus datasets, HBV and HCV, we assess if computational methods for molecular species delimitation that are typically applied to barcoding biodiversity studies can also be successfully deployed for hepatitis virus classification. For comparison, we also used ABGD, a tool that in contrast to other distance methods attempts to automatically identify the barcoding gap using pairwise genetic distances for a set of aligned input sequences.Results—DiscussionWe found that the mPTP species delimitation tool identified even without adapting its default parameters taxonomic clusters that either correspond to the currently acknowledged genotypes or to known subdivision of genotypes (subtypes or subgenotypes). In the cases where the delimited cluster corresponded to subtype or subgenotype, there were previous concerns that their status may be underestimated. The clusters obtained from the ABGD analysis differed depending on the parameters used. However, under certain values the results were very similar to the taxonomy and mPTP which indicates the usefulness of distance based methods in virus taxonomy under appropriate parameter settings. The overlap of predicted clusters with taxonomically acknowledged genotypes implies that virus classification can be successfully automated.

DNA barcode and phylogenetic study of the tribe Desmodieae (Fabaceae) in Korea

본 연구에서는 DNA 바코드, 즉 엽록체 DNA의 rbcL, matK와 핵 리보솜 DNA의 ITS 염기서열을 이용하여 한국산 ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì¡± 식물들의 ì¢ ì‹ë³„ì„ 실시하였다. 총 5개속 25분류군(총 75개체)의 식물에 대한 염기서열이 ë°í˜€ì¡Œê³ , DNA 바코드 구간 조합에 따라 neighbor-joining 계통수들이 작성되었다. ê·¸ ê²°ê³¼, ì¢ ì‹ë³„ë¥ ì€ DNA 바코드 3개의 구간을 모두 사용하였을 때 가장 높게 나타났다(72%). rbcL+matK+ITS 계통수에서 본 족의 두 개의 아족과 다섯 속들은 단계통군으로 ìœ ì§‘ë˜ì—ˆë‹¤. ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì•„ì¡±êµ°ì—ì„œ 도둑놈의 ê°ˆê³ ë¦¬ì†ê³¼ ê°ˆê³ ë¦¬ì†ì´ 된장풀속보다 더 가깝게 묶였다. ê°ˆê³ ë¦¬ì†êµ°ì—ì„œ í°ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ëŠ” ê°œë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ 복합체의 자매군으로 í˜•ì„±ë˜ì—ˆê³ , ê°œë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì˜ ì¢ ë‚´ë¶„ë¥˜êµ°ë“¤ì€ 각각 단계통을 형성하였다. 특히 í˜•íƒœì  변이로 인해 ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì˜ ë³€ì¢ ë˜ëŠ” ê°œë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì™€ì˜ 중간형으로 인식된 바 있는 ê¸´ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ëŠ” ê°œë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì™€ 가장 가깝게 묶였다. 한편 ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì˜ ë³€ì¢ ì´ë‚˜ ì´ëª ìœ¼ë¡œ 인식되어온 ì• ê¸°ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ëŠ” ë³€ì¢ ìœ¼ë¡œ 판단되었다. 싸리아족군의 경우, 매듭풀속 분류군들은 계통수상에서 각각 단계통으로 지지되었으나, 싸리속의 식물 16분류군 중 9분류군만 ì¢ ì‹ë³„ì´ 가능하였다. 특히 ì‹¸ë¦¬ì†ë‚´ë• ë¹„ìˆ˜ë¦¬ì ˆë³´ë‹¤ ì‹¸ë¦¬ì ˆì—ì„œ 낮은 ì¢ íŒë³„ 해상능이 나타났다(ì‹¸ë¦¬ì ˆ=28.5%, ë¹„ìˆ˜ë¦¬ì ˆ=77.8%). 싸리속 식물 중에 비수리는 계통수상에서 í˜•íƒœì ìœ¼ë¡œ ìœ ì‚¬í•œ 자주비수리와 확연히 구분되었다. ìž¡ì¢ ìœ¼ë¡œë„ 인식된 바 있는 해변싸리와 청비수리 중, 해변싸리는 ë ë¦½ì¢ ìœ¼ë¡œ 판단되었다. 반면 청비수리는 ìž¡ì¢ ì—¬ë¶€ë¥¼ íŒë‹¨í• ìˆ˜ëŠ” 없었지만, ë• ë¹„ìˆ˜ë¦¬ì™€ 근연 관계에 있는 것으로 사료된다. 주요어: ë„ë‘‘ë†ˆì˜ê°ˆê³ ë¦¬ì¡±, ì¢ ì‹ë³„, DNA 바코드 rbcL, matK, ITS

A DNA barcoding study of seven cichlid species from southern Africa reveals their phylogenetic relationships

This study of three Namibian fish species (two endemic Thoracochromis and Tilapia guinasana) and Pseudocrenilabrus philander reveals their monophyly. These species (and Sandelia capensis) have never been DNA barcoded and their phylogenetic relationships with two Tilapia species is shown. Average K2P intraspecies distances between this fish DNA barcoding study ranged from zero (Thoracochromis albolaris) to 10% for the outgroup. There are approximately 17 Thoracochromis (Greenwood 1979) species in Africa, with only two in southern Africa (Skelton 2001). Both occur in the Cunene River System (at the border of Angola and Namibia). Thoracochromis albolaris (Trewavas and Thys van den Audenaerde 1969) – abbreviated as Tha – prefers rocky areas, whereas Thoracochromis buysi (Penrith 1970) – Thb – prefers sandy habitats. Pseudocrenilabrus philander (Weber 1897) – Pp – is a widely distributed species in Africa (Skelton 2001). Tilapia guinasana Trewavas 1936 – Tg – is endemic to Namibia, endangered and confined to Lake Guinas. These are very colourful fish (e.g. Tg varies in colour combinations from white to blue, yellow, black, olive green and some even have red fins).

Evidence of Intra-individual SSU Polymorphisms in Dark-spored Myxomycetes (Amoebozoa)

The nuclear small subunit rRNA gene (SSU or 18S) is a marker frequently used in phylogenetic and barcoding studies in Amoebozoa, including Myxomycetes. Despite its common usage and the confirmed existence of divergent copies of ribosomal genes in other protists, the potential presence of intra-individual SSU variability in Myxomycetes has never been studied before. Here we investigated the pattern of nucleotide polymorphism in the 5' end fragment of SSU by cloning and sequencing a total of 238 variants from eight specimens, each representing a species of the dark-spored orders Stemonitidales and Physarales. After excluding singletons, a relatively low SSU intra-individual variability was found but our data indicate that this might be a widely distributed phenomenon in Myxomycetes as all samples analyzed possessed various ribotypes. To determine if the occurrence of multiple SSU variants within a single specimen has a negative effect on the circumscription of species boundaries, we conducted phylogenetic analyses that revealed that clone variation may be detrimental for inferring phylogenetic relationships among some of the specimens analyzed. Despite that intra-individual variability should be assessed in additional taxa, our results indicate that special care should be taken for species identification when working with closely related species.

Review and Interpretation of Trends in DNA Barcoding

Interpretations and analytical practices surrounding DNA barcoding are reviewed from a compilation of 3,756 papers (as of December 31, 2018) with “DNA Barcode” in the title since 2004. By examining the practice of DNA barcoding in natural history and biodiversity science over this period, we explore the extent to which its purposes, premises, rationale and application have evolved. The number of studies involving identification, taxonomic decisions and the discovery of cryptic species has driven the publication of DNA barcode studies overall. Forensic studies and papers on biological conservation involving DNA barcodes have tracked the ensemble number of studies but rose sharply in 2017. Although neighbor-joining and graphic (tree-based) criteria for species delimitation have been preeminent, analytical paradigms have diversified slightly following the growing availability of tools in the Barcode of Life Database (BoLD). We conclude that the paradigms of DNA barcoding data are likely to persist and, in groups such as Lepidoptera, DNA barcoding has become a widely used tool in taxonomic science. The degree to which systematists will avail themselves of tools for extracting diagnostic data from barcodes remains to be seen.

Novel Data on the Taxonomic Diversity, Distribution, and Host Plants of Leafmining Moths of the Family Gracillariidae (Lepidoptera) in Siberia, Based on DNA Barcoding

The Gracillariidae fauna of Siberia, the region that occupies almost half of the territory of the Russian Federation, remains poorly studied. During a DNA barcoding study of Gracillariidae in Siberia, based on analysis of larvae and pupae collected from their leaf mines on woody plants, we identified 41 species. Three gracillariids were identified only to genus: Caloptilia sp. (host plant Prunus padus), Parornix sp. (Malus sp.), and Phyllonorycter sp. (Crataegus sp.), representing poorly studied or undescribed species. Six species are reported here for the first time for Siberia: Callisto insperatella (from Novosibirsk and Tomsk provinces), Caloptilia alnivorella (Buryatia), Phyllonorycter ermani (Irkutsk Province), Ph. lantanella (Novosibirsk Province), Ph. pumilae (Omsk and Irkutsk provinces), and Ph. viciae (Krasnoyarsk Territory). Parornix pfaffenzelleri, found in Khakassia, is reported as a species new to Russia. Other 15 gracillariid species previously known from Siberia were recorded in new administrative regions. The invasive lime leafminer Phyllonorycter issikii was first documented in Tomsk Province and Krasnoyarsk Territory in 2017 and 2018, respectively. Seven new gracillariid-host plant associations were found: Parectopa ononidis on Lupinaster pentaphyllus, Sauterina hofmanniella on Lathyrus gmelinii, Caloptilia stigmatella on Salix kochiana, Callisto insperatella on Prunus virginiana, Parornix scoticella on Amelanchier sp., Phyllonorycter ermani on Alnus alnobetula subsp. fruticosa, and Phyllonorycter viciae on Vicia unijuga. The Gracillariidae fauna of Siberia has 51% similarity with that of European Russia and only 38% similarity with that of the Russian Far East.

Description of a new small-sized Synodontis species (Siluriformes: Mochokidae) that is important for local subsistence fisheries in the middle Lufira (upper Congo River, DR Congo).

Synodontis denticulatus sp. nov. is an endemic from the middle Lufira Basin and its associated tributaries and lakes. The species shows close morphological resemblance to Synodontis greshoffi and Synodontis unicolor, which are widespread Congo Basin and Bangweulu-Mweru endemic species, respectively. However, it differs from both S. greshoffi and S. unicolor by its non-villous skin (v. villous skin), strong and numerous serrations on the posterior margin of the dorsal spine (v. weak and fewer serrations), weak and few serrations on the posterior margin of the pectoral spine (v. strong and numerous serrations), relatively short maxillary barbels (v. long) and its small maximum standard length (89.1 mm LS v. 148.0 and 190.7 mm LS respectively). A DNA barcoding study (coI, mtDNA) revealed that S. denticulatus forms a distinct genetic clade with a genetic distance of 2.18% with S. greshoffi and 0.84% with S. unicolor. Synodontis denticulatus is caught regularly and abundantly as a by-catch in the gillnet fisheries in the middle Lufira lakes. Owing to its small overall size and large bony head, the species has usually no real commercial value but is an important food fish for the fishermen's families.

DNA barcode and minibarcode identification of freshwater fishes from Cerrado headwater streams in Central Brazil.

The extraordinary species diversity of the Neotropical freshwater fish fauna is world renown. Yet, despite rich species diversity, taxonomic and genetic resources for its Cerrado ichthyofauna remain poorly developed. We provide a reference library of 149 DNA barcodes for 39 species/lineages of Cerrado headwater stream fishes from the Brazilian Distrito Federal and nearby areas and test the utility of distance-based criteria, tree-based criteria and minibarcodes for specimen identification. Mean Kimura 2-parameter genetic distances within species to orders ranged 1·8-12·1%. However, mean intraspecific v. congeneric-interspecific distances (0·9-1·3%) overlapped extensively and distance-based barcoding failed to achieve correct identifications due to c. 4-12·1% error rates and 19·5% ambiguous identifications related to the presence of singletons. Overlap was reduced and best-match success rates improved drastically to 83·5% when Characidium barcodes representing potential misidentifications or undescribed species were removed. Tree-based monophyly criteria generally performed similarly to distance methods, correctly differentiating up to c. 85% of species/lineages despite neighbour-joining and Bayesian tree errors (random lineage-branching events, long-branch attraction). Five clusters (Ancistrus aguaboensis, Characidium spp., Eigenmannia trilineata, Hasemania hanseni and Hypostomus sp. 2) exhibited deep intraspecific divergences or para-/polyphyly and multiple Barcode Index Number assignments indicative of putative candidate species needing taxonomic re-examination. Sliding-window analyses also indicated that a 200 bp minibarcode region performed just as well at specimen identification as the entire barcode gene. Future DNA barcoding studies of Distrito Federal-Cerrado freshwater fishes will benefit from increased sampling coverage, as well as consideration of minibarcode targets for degraded samples and next-generation sequencing.

DNA barcodes fail to accurately differentiate species in Hawaiian plant lineages

AbstractDNA barcoding has been largely successful in differentiating animal species, but the most effective loci and evaluative methods for plants are still debated. Floras of young, oceanic islands are a challenging test of DNA barcodes, because of rapid speciation, high incidence of hybridization and polyploidy. We used character-based, tree-based and genetic distance-based methods to test DNA barcoding of 385 species of native Hawaiian plants constituting 20 lineages at the nuclear ITS(2) locus, nine lineages at each of the plastid loci trnH-psbA and rbcL, eight lineages at the plastid locus matK and four lineages with concatenated data. We also incorporated geographical range information and tested if varying sample sizes within a lineage influenced identification success. Average discrimination success was low (22% maximum) with all methods of analysis across all loci. The character-based method generally provided the highest identification success, there were limited benefits from incorporating geographical data and no relationship between number of species sampled in a lineage and identification success was found. Percentages of identification success are the lowest reported in a DNA barcoding study of comparable scale, and multi-species groups that radiated in the Hawaiian archipelago probably cannot be identified based on current DNA barcoding loci and methodologies.

Sharpening species boundaries in the Micarea prasina group, with a new circumscription of the type species M. prasina

ABSTRACTMicarea is a lichenized genus in the family Pilocarpaceae (Ascomycota). We studied the phylogeny and reassessed the current taxonomy of the M. prasina group. We focused especially on the taxonomic questions concerning the type species M. prasina and, furthermore, challenges concerning type specimens that are too old for successful DNA barcoding and molecular studies. The phylogeny was reconstructed using nuc rDNA internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS), mitochrondrial rDNA small subunit (mtSSU), and replication licensing factor MCM7 gene from 31 species. Fifty-six new sequences were generated. The data were analyzed using maximum parsimony and maximum likelihood methods. The results revealed four undescribed, well-supported lineages. Three lineages represent new species described here as M. fallax, M. flavoleprosa, and M. pusilla. In addition, our results support the recognition of M. melanobola as a distinct species. Micarea fallax is characterized by a vivid to olive green thallus composed of aggregated granules and whitish or brownish apothecia sometimes with grayish tinge (Sedifolia-gray pigment).Micarea flavoleprosa has a thick, wide-spreading yellowish green, whitish green to olive green sorediate thallus and lacks the Sedifolia-gray pigmentation. The species is mostly anamorphic, developing apothecia rarely. Micarea melanobola is characterized by a pale to dark vivid green granular thallus and darkly pigmented apothecia (Sedifolia-gray). Micarea pusilla is characterized by a whitish green to olive green thinly granular or membranous thallus, numerous and very small whitish apothecia lacking the Sedifolia-gray pigment, and by the production of methoxymicareic acid. Micarea fallax, M. flavoleprosa, and M. melanobola produce micareic acid. The reliability of crystalline granules as a character for species delimitation was investigated and was highly informative for linking the old type specimen of M. prasina to fresh material.

Discovery of Aphis ruborum (Hemiptera: Aphididae) and Aphelinus varipes (Hymenoptera: Aphelinidae) on Cultivated Strawberry in Mississippi, USA.

An adventive aphid and novel host-parasitoid association from cultivated strawberry (Fragaria × ananessa Duch. cv. Chandler; Fragaria × ananessa Duch. cv. Camarosa) in Mississippi, USA are reported herein. The aphid, first detected in high tunnel cultivation, was found predominately on newly emerged, not fully developed leaflets of daughter plants in the Fall of 2016. By 2017, aphids and their associated mummies were observed on fully developed leaflets on mother plants of both cultivars. The aphid was identified as Aphis ruborum (Börner & Schilder) using morphology and DNA barcoding studies. In addition, DNA barcoding identified parasitoid adults emerging from aphid mummies as two cryptic species, Aphelinus varipes (Foerster) and Aphelinus albipodus Hayat and Fatima. Occurrence of A. ruborum in Mississippi represents a new state record and the eastern-most established record in the United States. The A. ruborum - A. varipes or A. albipodus host-parasitoid association is reported for the first time anywhere in the world.

Intragenomic internal transcribed spacer 2 variation in a genus of parasitoid wasps (Hymenoptera: Braconidae): implications for accurate species delimitation and phylogenetic analysis.

A recent DNA barcoding study of Australian microgastrines (Hymenoptera: Braconidae) sought to use next-generation sequencing of the cytochrome c oxidase subunit 1 (COI) barcoding gene region, the wingless (WG) gene and the internal transcribed spacer 2 (ITS2) to delimit molecular species in a highly diverse group of parasitic wasps. Large intragenomic distances between ITS2 variants, often larger than the average interspecific variation, caused difficulties in using ITS2 for species delimitation in both threshold and tree-based approaches, and the gene was not included in the reported results of the previous DNA barcoding study. We here report on the intragenomic, and the intra- and interspecies, variation in ITS2in the microgastrine genus Diolcogasterto further investigate the value of ITS2as a marker for species delimitation and phylogenetics of the Microgastrinae. Distinctive intragenomic variant patterns were found in different species of Diolcogaster, with some species possessing a single major variant, and others possessing many divergent variants. Characterizing intragenomic variation of ITS2is critical as it is a widely used marker in hymenopteran phylogenetics and species delimitation, and large intragenomic distances such as those found in this study may obscure phylogenetic signal.

Barcoding the Neotropical freshwater fish fauna using a new pair of universal COI primers with a discussion of primer dimers and M13 primer tails.

Designing primers for DNA barcoding is a significant challenge for the rich Neotropical fish fauna, which is comprised of ∼6000 species. Previously, researchers required multiple pairs of PCR primers or primer cocktails to obtain standard COI (i.e., mitochondrial cytochrome c oxidase subunit I) barcode sequences from assemblages of freshwater fish in this region. To simplify DNA barcoding and metabarcoding studies of Neotropical freshwater fish, we present a new pair of COI primers, which have yielded high quality barcodes across six teleost orders-Characiformes, Cichliformes, Cyprinodontiformes, Gymnotiformes, Siluriformes, and Synbranchiformes-native to South America. Following previous fish barcoding studies, we also tailed our primers with M13 forward and reverse primers to facilitate the DNA sequencing process. Although this practice generates primer dimers, we obtained complete and high quality COI barcode sequences for all samples. We discuss the problem of primer dimers and suggest strategies for neutralizing their influence on data quality.

Revisiting theLepetodrilus elevatusspecies complex (Vetigastropoda: Lepetodrilidae), using samples from the Galápagos and Guaymas hydrothermal vent systems

The current distribution ranges of vent species result from the complex tectonic history of oceanic ridges. A growing number of DNA barcode studies report the presence of cryptic species across geological discontinuities that offset ridge systems and have gradually helped to draw a more precise picture of the historical migration pathways of vent fauna. We reexamined the phylogeny of species within the Lepetodrilus elevatus complex along the East Pacific Rise (EPR) ridge system in the light of new samples from the Galapagos Rift and the Guaymas Basin. Our analyses of mitochondrial cytochrome c oxidase subunit I gene sequences, coupled with morphological data, highlight the occurrence of a distinct lineage along the Galapagos Rift and offer new insight into the current distribution range of this species complex. Due to the absence of clear morphological diagnostic criteria and the potential overlap of these lineages at key locations, we recommend reassigning the taxon L. galriftensis to the subspecies level and maintaining the name L. elevatus for all clades along the EPR/Galapagos Rift system.

The DNA barcoding of Mediterranean combtooth blennies suggests the paraphyly of some taxa (Perciformes, Blenniidae).

A dataset including novel and publicly available mtDNA COI sequences of 14 Mediterranean combtooth blenny species belonging to nine genera was assembled in order to provide a reference dataset for DNA barcoding studies. Some inconsistencies in the current taxonomy of some genera were observed. In particular, the monophyly of the genera Parablennius and Salaria is not supported by the present dataset and the absence of reciprocal monophyly between the morphospecies Salaria basilisca and S. pavo questions their status and stresses the need of a revision of the genus Salaria.

New data and hypotheses on the invasiveness, habitat selection, and ecological role of the limicolous earthworm Sparganophilus tamesis Benham, 1892

A recent molecular barcode study certified the conspecificity of mutually distant European populations of the limicolous earthworm Sparganophilus tamesis Benham, 1892 with the most widespread species of Sparganophilus in North America, and that all the analysed European worms descend from a recent introduction, probably via one or more point-source events with subsequent dispersion. We present the first records from Belgium and the Atlantic coast of southern France, and further genetic evidence confirming the species’ high dispersal abilities and historical movement within and across European drainage boundaries, as well as its capacity to establish in different types of aquatic ecosystems, including oligotrophic habitats. Experimental evidence suggests that the activity of Sparganophilus combined with that of oxygen-releasing plants, such as Lobelia dortmanna, may enhance the mobilization of refractory N pools within oligotrophic sediments, and, therefore, act as facilitator for both the macrophyte and the microbial communities in the rhizosphere. © 2018 E. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart, Germany.

Using next-generation sequencing to improve DNA barcoding: lessons from a small-scale study of wild bee species (Hymenoptera, Halictidae)

The parallel sequencing of targeted amplicons is a scalable application of next-generation sequencing (NGS) that can advantageously replace Sanger sequencing in certain DNA barcoding studies. It can be used to sequence different PCR products simultaneously, including co-amplified products. Here, we explore this approach by simultaneously sequencing five markers (including the DNA barcode and a diagnostic marker of Wolbachia) in 12 species of Halictidae that were previously DNA barcoded using Sanger sequencing. Consensus sequences were obtained from fresh bees with success rates of 74–100% depending on the DNA fragment. They improved the phylogeny of the group, detected Wolbachia infections (in 8/21 specimens) and characterised haplotype variants. Sequencing cost per marker and per specimen (11.43 €) was estimated to decrease (< 5.00 €) in studies aiming for a higher throughput. We provide guidelines for selecting NGS or Sanger sequencing depending on the goals of future studies.