The gregarious species of Apanteles that attack cactus-feeding phycitine moths (Lepidoptera : Pyralidae) from Argentina are identified using DNA barcodes and morphological data. Sequences of specimens from 10 different populations were generated. Corrected genetic divergences showed two clusters of specimens separated by COI divergences higher than 6%. Our morphological examinations were congruent with the molecular evidence and therefore two species were confidently identified: Apanteles alexanderi Brèthes and a new species, Apanteles opuntiarum Martínez & Berta, sp. nov., which we describe and illustrate. Despite these two taxa being considered to represent a single polyphagous species for at least 50 years, they can be easily distinguished by the length of their ovipositor sheaths. A key to the species of microgastrine parasitoids of cactus-feeding Lepidoptera in South America is provided. The recognition of the new species reveals a narrower host range for the species involved in this system, making it necessary to re-evaluate their potential as biocontrol agents of the prickly pear moth.

Interspecific hybridisation is a rare but widespread phenomenon identified as a potential complicating factor for the identification of species through DNA barcoding. Hybrids can, however, also deceive morphology-based taxonomy, resulting in the description of invalid species based on hybrid specimens. As the result of an unexpected case of discordance between barcoding results and current morphology-based taxonomy, we discovered an example of such a hybrid ‘species’ in hawkmoths. By combining barcodes, morphology and a nuclear marker, we show that Gnathothlibus collardi Haxaire, 2002 is actually an F1 hybrid between two closely related species that co-occur on Tahiti. In accordance with the International Code of Zoological Nomenclature, the taxon G. collardi is thus invalid as a species. This study demonstrates the potential of DNA barcodes to detect overlooked hybrid taxa. With the growth of sequence libraries, we anticipate that more unsuspected hybrid species will be detected, particularly among those taxa that are very rare, such as those known from only the type specimen.

Molecular identification systems depend on genetic diversity based on a short mitochondrial DNA fragment being markedly lower within than between species to infer identification of specimens. Our analyses show that both COI and CytB fragments can successfully distinguish most Ruspolia species. R. jezoensis was synonymised with R. dubia, and R. liangshanensis may be recently separated from R. dubia. R. indica, often called Euconocephalus indicus, occupied a well supported position within Ruspolia, and should be transferred into Ruspolia. The individuals of R. lineosa from Yunnan formed a monophyletic group at the base of the R. lineosa clade, and the Kimura 2-parameter distance between it and other R. lineosa individuals was 0.065 for COI and 0.069 for CytB, which may be the result of isolated or remote populations. However, for recently diverged taxa R. liangshanensis and R. dubia, straightforward application of barcoding rules may prove problematic without morphological, behavioural or ecological data.

Solenopsis is a widespread ant genus and the identification of its species is notoriously difficult. Hence, investigation of their distribution along elevational gradients is challenging. Our aims were (1) to test the complementarity of the morphological and DNA barcoding approaches for Solenopsis species identification, and (2) to assess species diversity and distribution along an altitudinal gradient in the Ecuadorian Andes. Ants were collected in five localities between 1000 and 3000 m above sea level. In total, 24 morphospecies were identified along the gradient and 14 of them were barcoded. Seven morphospecies were confirmed by the molecular approach. Three others, occurring sympatrically and possessing clear diagnostic characters, showed low genetic divergence. Representatives of a further four morphospecies were split into nine clusters by COI and nuclear wingless genetic markers, suggesting the existence of cryptic species. Examination of gynes revealed potential diagnostic characters for morphological discrimination. Solenopsis species were found up to an altitudinal record of 3000 m. Most morphospecies (20 of 24) were found at a single elevation. Our results suggest a high species turnover along the gradient, and point to the use of morphological and DNA barcoding approaches as necessary for differentiating among Solenopsis species.

Collembola is one of the major functional groups in soil as well as a model taxon in numerous disciplines. Therefore the accurate identification of specimens is critical, but could be jeopardised by cases of cryptic diversity. Several populations of six well characterised species of springtails were sequenced using the COI barcode fragment as a contribution to the global Collembola barcoding campaign. Each species showed high intraspecific divergence, comparable to interspecific sequence divergence values observed in previous studies and in 10 congeneric species barcoded here as a reference. The nuclear marker, 28S, confirmed all the intraspecific lineages found with COI, supporting the potential specific status of these entities. The implications of this finding for taxonomy and for disciplines relying on species names, such as evolution and ecology, are discussed.

Biodiversity of tropical Saturniidae, as measured through traditionally described and catalogued species, strongly risks pooling cryptic species under one name. We examined the DNA barcodes, morphology, habitus and ecology of 32 ‘well known’ species of dry forest saturniid moths from Area de Conservacion Guanacaste (ACG) in north-western Costa Rica and found that they contain as many as 49 biological entities that are probably separate species. The most prominent splitting of traditional species – Eacles imperialis, Automeris zugana, Automeris tridens, Othorene verana, Hylesia dalina, Dirphia avia, Syssphinx molina, Syssphinx colla, and Syssphinx quadrilineata – is where one species was believed to breed in dry forest and rain forest, but is found to be two biological entities variously distinguishable by DNA barcodes and morphology, habitus, and/or microecological distribution. This implies that ‘standard’ biological information about each traditional species may be an unconscious mix of interspecific information, and begs renewed DNA barcoding, closer attention to so-called intraspecific variation, and increased museum collection and curation of specimens from more individual and ecologically characterised sites – as well as eventually more species descriptions. Simultaneously, this inclusion of sibling species as individual entities in biodiversity studies, rather than pooled under one traditional name, reduces the degree of ecological and evolutionary generalisation perceived by the observer.

This study examines the genetic data coverage and availability in the Barcode of Life Database (BOLD), versions 2.5 and 3.0, and GenBank for the 88 invasive insects listed in the Global Invasive Species Database ( http://www.issg.org). No data are recorded in either BOLD or GenBank for seven of those species. As a dedicated repository of curated barcode data BOLD is either missing data or contains inaccessible private data for 37 (42%) of the species while no data are available in GenBank for nine (8%) of the species. An evaluation of the Barcode Identification Number (BIN) scheme in BOLD ver. 3.0 was also evaluated and in 41% of cases the BIN contained more than one species. This essentially arose due to the 1% delimitation thresholds associated with the BINs and would result in misidentifications. Overall, more information is available from GenBank for the 88 invasive species listed on the Global Invasive Species Database, but quality checking is required to ensure that the data extracted from GenBank are of sufficient quality to make it useful. The implications of these results are discussed, with investment in parallel data silos suggested to be both costly and potentially an inefficient use of resources that may lead to loss of data if the means needed to maintain these databases become unavailable.

Carrion-breeding Sarcophagidae (Diptera) can be used to estimate the post-mortem interval in forensic cases. Difficulties with accurate morphological identifications at any life stage and a lack of documented thermobiological profiles have limited their current usefulness. The molecular-based approach of DNA barcoding, which utilises a 648-bp fragment of the mitochondrial cytochrome oxidase subunit I gene, was evaluated in a pilot study for discrimination between 16 Australian sarcophagids. The current study comprehensively evaluated barcoding for a larger taxon set of 588 Australian sarcophagids. In total, 39 of the 84 known Australian species were represented by 580 specimens, which includes 92% of potentially forensically important species. A further eight specimens could not be identified, but were included nonetheless as six unidentifiable taxa. A neighbour-joining tree was generated and nucleotide sequence divergences were calculated. All species except Sarcophaga (Fergusonimyia) bancroftorum, known for high morphological variability, were resolved as monophyletic (99.2% of cases), with bootstrap support of 100. Excluding S. bancroftorum, the mean intraspecific and interspecific variation ranged from 1.12% and 2.81–11.23%, respectively, allowing for species discrimination. DNA barcoding was therefore validated as a suitable method for molecular identification of Australian Sarcophagidae, which will aid in the implementation of this fauna in forensic entomology.

Recent studies have suggested that some resident Antarctic biota are of ancient origin and may have been isolated for millions of years. The phylum Tardigrada, which is part of the Antarctic terrestrial meiofauna, is of particular interest due to an impressive array of biochemical abilities to withstand harsh environmental conditions. Tardigrades are one of the few widespread Antarctic terrestrial animals that have the potential to be used as a model for evolution and biogeography on the Antarctic continent. We isolated 126 individual tardigrades from four geographically isolated soil samples from two remote nunataks in the Sør Rondane Mountains, Dronning Maud Land, Antarctica. We examined genetic variation among individuals utilising three gene regions: cytochrome c oxidase subunit I gene (COI), 18S rDNA (18S), and the wingless (Wg) gene. Comparison of sequences from worldwide and Antarctic tardigrades indicated long-term survival and isolation over glacially dominated periods in ice-free habitats in the Sør Rondane Mountains.

A fragment of the cytochrome c oxidase subunit I (COI) gene has been used increasingly for species identification and discovery in eukaryotes. However, amplifying COI has proven difficult, or even impossible, in some taxa due to non-homology between the universal primers and the target DNA region. Among the most problematic animal groups is Serpulidae (Annelida). These sedentary marine animals live in self-secreted calcareous tubes and many of them, especially of the genus Hydroides, are economically important reef-builders, foulers, and biological invaders. We developed novel taxon-specific primers for amplifying COI from Hydroides, and for the first time generated 460-bp COI sequences from 11 of 14 species attempted. Average Kimura-2-parameter interspecific sequence distance (26.2%) was >60 times greater than the average intraspecific distance (0.43%), indicating that the COI gene is effective for species delimitation in Hydroides. Although applicability of the new primers for a wide range of serpulids needs to be tested, barcoding of Hydroides is now on its way from impossible to difficult. We anticipate that COI barcoding will provide a modern species identification tool and, combined with other molecular markers, yield important insights in phylogeny and evolutionary ecology of this large and important genus.

DNA barcoding studies to elucidate the evolutionary and dispersal history of the current populations of Nautilus pompilius allow us to develop a greater understanding of their biology, their movement and the systematic relationships between different groups. Phylogenetic analyses were conducted on Australian N. pompilius, and COI sequences were generated for 98 discrete accessions. Sequences from samples collected across the distribution were sourced from GenBank and included in the analyses. Maximum likelihood revealed three distinct clades for N. pompilius: (1) populations sourced from west Australia, Indonesia and the Philippines; (2) populations collected from east Australia and Papua New Guinea; (3) western Pacific accessions from Vanuatu, American Samoa and Fiji, supporting previous findings on the evolutionary divergence of N. pompilius. A minimum spanning tree revealed 49 discrete haplotypes for the 128 accessions, from a total of 16 discrete sampling locations. Population similarity reflects oceanic topographic features, with divergence between populations across the N. pompilius range mirroring geographical separation. This illustrates the success of DNA barcoding as a tool to identify geographic origin, and looks to the future role of such technology in population genetics and evolutionary biology.

The taxonomic status of the species Clibanarius sclopetarius (Herbst, 1796) and Clibanarius vittatus (Bosc, 1802), which have sympatric biogeographical distributions restricted to the western Atlantic Ocean, is based only on differences in the colour pattern of the walking legs of adults. Their morphological similarity led to the suggestion that they be synonymised. In order to investigate this hypothesis, we included species of Clibanarius Dana, 1892 in a molecular phylogenetic analysis of partial sequences of the mitochondrial 16S rDNA gene and the COI barcode region. In addition, we combined the molecular results with morphological observations obtained from several samples of these two species. The genetic divergences of the 16S rDNA and COI sequences between C. sclopetarius and C. vittatus ranged from 4.5 to 5.9% and 9.4 to 11.9%, which did not justify their synonymisation. Differences in the telson morphology, chela ornamentation, and coloration of the eyestalks and antennal peduncle provided support for the separation of the two species. Another interesting result was a considerable genetic difference found between populations of C. vittatus from Brazil and the Gulf of Mexico, which may indicate the existence of two homonymous species.

Marine wood-boring teredinids, some of the most destructive wood borers in the sea, are a particularly difficult group to identify from morphological features. While in most bivalve species shell features are used as diagnostic characters, in the teredinids shell morphology shows high intraspecific variation and thus identification is based almost entirely on the morphology of the pallets. In the present study we aimed at improving ‘taxonomic resolution’ in teredinids by combining morphological evidence with mitochondrial and nuclear DNA sequences, respectively Cytochrome c oxidase subunit I and small subunit rRNA 18S gene, to generate more rigorous and accessible identifications.

DNA barcodes of Atlantic and Mediterranean populations of Lyrodus pedicellatus diverged by ∼20%, suggesting cryptic species in the morphospecies L. pedicellatus. The low intraspecific divergence found in barcodes of specimens of Nototeredo norvagica (0.78%) confirms that Atlantic and Mediterranean forms of N. norvagica, the latter sometimes reported as Teredo utriculus, are the same species. Teredothyra dominicensis was found for the first time in the Mediterranean. A match was obtained between our 18S sequences and sequences of T. dominicensis from Netherlands Antilles, confirming that T. dominicensis in the Mediterranean is the same species that occurs in the Caribbean. There were differences in 18S sequences between Bankia carinata from the Mediterranean and Caribbean, which may indicate cryptic species.

The eastern Australian aquifers remain mostly unexplored; however, recent surveys suggest that there could be substantial levels of subterranean biodiversity hidden in these aquifers. Groundwater fauna (stygofauna) is often characterised by short-range endemism. Furthermore, high levels of cryptic species, and lack of formal taxonomic descriptions and taxonomic expertise for many of the groups demand innovative approaches for assessing subterranean biodiversity. Here we evaluate the potential of using DNA barcoding as a rapid biodiversity assessment tool for the subterranean groundwater fauna of New South Wales, Australia. We experienced low amplification success using universal and more taxon-specific primers for PCR amplification of the barcoding gene (COI) in a range of crustacean stygofauna. Sequence comparisons of the most commonly used COI universal primers in selected crustacean taxa revealed high levels of variability. Our results suggest that successful amplification of the COI region from crustacean stygofauna is not straightforward using the standard ‘universal’ primers. We propose that the development of a multiprimer (taxon specific) and multigene approach for DNA barcode analyses, using next-generation sequencing methodologies, will help to overcome many of the technical problems reported here and provide a basis for using DNA barcoding for rapid biodiversity assessments of subterranean aquatic ecosystems.

The sequencing of the crustacean collection of the MNHN, Paris, constitutes a promising yet very challenging barcoding project. For the collection’s crustacean specimens preserved in ethanol, some of which were collected up to 40 years ago, the conventional COI barcoding procedure of amplification with Folmer primers failed for more than half of the specimens (58%, n = 1920). We hypothesised that this failure may have been due to incompatible mismatches between the crustaceans targeted and the Folmer primer sequences and/or the amount of degradation of the DNA extracted from museum specimens. The comparison of the Folmer primers against the COI sequences from GenBank complete decapod mitochondrial genomes revealed that the annealing regions were, in fact, rather conserved, suggesting that the amplification failures were due more likely to the low quality of the DNA isolated. Using an alignment of all available decapod sequences we designed two internal primers in the middle of the barcoding COI region and also selected two additional external primers to be used as alternative to the standard Folmer primers. Using a two-overlapping-fragments amplification strategy and different primer combinations, our new protocol significantly increased the amplification success rate of the collection material from 42% with the Folmer primers to 84%, recovering an additional 364 complete barcodes and 443 minibarcodes (i.e. fragments of less than 400 base pairs), and expanding the species coverage from 254 to 397 barcoded crustaceans.