DNA barcoding reveals the occurrence of cryptic species in host-associated population of Conogethes punctiferalis (Lepidoptera: Crambidae)

Conogethes punctiferalis (Guénee) is a critical pest that commonly infests castor (Ricinus communis Linnaeus) and cardamom (Elettaria cardamomum Maton) in India. The moths of both castor and cardamom appear to be similar in wing pattern and color. However, the results of behavioral studies elicited a doubt that there may be differences in terms of host specialization. In the present study, we conducted morphological studies and DNA barcode analyses using cytochrome oxidase I gene, which unraveled the mystery of C. punctiferalis breeding on castor and cardamom. The differences in male aedeagus and female bursae were prominent, yet, not sufficient enough to say that they are different species. The results showed high haplotype diversity (0.817 ± 0.073) and nucleotide diversity (0.0285 ± 0.002) in C. punctiferalis. In addition, topologies of neighbor-joining trees indicate that Conogethes sp. breeding on castor belongs to C. punctiferalis while those on cardamom are of a separate clade. Further genetic analysis revealed significant genetic differentiations among the two sampled populations, reflecting limited gene flow. Neutrality tests and mismatch distributions showed population expansion in C. punctiferalis, while the results of an analysis of molecular variance (AMOVA) indicated the existence of significant genetic variation among the examined host races. Conclusively, analysis using mitochondrial DNA showed an amount of genetic divergence between the two host-associated populations compatible with cryptic species rather than host races.     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.     

DNA barcoding of metacestodes found in the Guerlinguetus ingrami (Rodentia: Sciuridae) reveals the occurrence of Hydatigera taeniaeformis sensu stricto (Cyclophyllidea: Taeniidae) in the Americas

The existence of cryptic species in the genus Hydatigera, cyclophillid cestodes, mainly of felids, was recently described based on molecular studies of parasites from Asia, Europe and Africa. However, the occurrence of H. taeniaeformis sensu stricto (s.s.), the species more widely distributed and with a presumed specificity for murid rodents as intermediate hosts, has not been formally described in Americas. In the present study, during necropsy of an Ingram's squirrel specimen, Guerlinguetus ingrami, found dead in the municipality of Belo Horizonte, Minas Gerais state, Brazil, strobilocerci were found in the liver. The metacestodes were subjected to morphological and molecular studies. Sequences of the COI barcode region were obtained and used for phylogenetic analyses. The morphology and measures of the rostellar hooks were compatible with the ones described for H. taeniaeformis s.s. This identification was confirmed by a molecular phylogenetic approach (96.2–99.7% similarity with isolates of the parasite from Europe and Asia). This is the first molecular confirmation of the existence of H. taeniaeformis s.s. on the American continent. Moreover, the involvement of sciurid rodents in the transmission of H. taeniaeformis s.s. is discussed here as a probable case of parasite spillover.     

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.     

DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation

DNA barcoding has revealed unrecognized species in several animal groups. In this study we have employed DNA barcoding to examine Hyalella, a taxonomically difficult genus of amphipod crustaceans, from sites in the southern Great Basin of California and Nevada, USA. We assessed the extent of species diversity using a species screening threshold (SST) set at 10 times the average intrapopulation cytochrome c oxidase subunit I (COI) haplotype divergence. Despite the fact that this threshold approach is more conservative in delineating provisional species than the phylogenetic species concept, our analyses revealed extraordinary levels of cryptic diversity and endemism. The SST discriminated two provisional species within Hyalella sandra, and 33 provisional species within Hyalella azteca. COI nucleotide divergences among these provisional species ranged from 4.4% to 29.9%. These results have important implications for the conservation of life in desert springs - habitats that are threatened as a result of groundwater over-exploitation.     

DNA barcoding highlights a cryptic species of grenadier Macrourus in the Southern Ocean

Although three species of the genus Macrourus are recognized in the Southern Ocean, DNA sequencing of the mitochondrial COI gene revealed four well-supported clades. These barcode data suggest the presence of an undescribed species, a conclusion supported by meristic and morphometric examination of specimens.     

From barcodes to genomes: extending the concept of DNA barcoding

DNA barcoding has had a major impact on biodiversity science. The elegant simplicity of establishing massive scale databases for a few barcode loci is continuing to change our understanding of species diversity patterns, and continues to enhance human abilities to distinguish among species. Capitalizing on the developments of next generation sequencing technologies and decreasing costs of genome sequencing, there is now the opportunity for the DNA barcoding concept to be extended to new kinds of genomic data. We illustrate the benefits and capacity to do this, and also note the constraints and barriers to overcome before it is truly scalable. We advocate a twin track approach: (i) continuation and acceleration of global efforts to build the DNA barcode reference library of life on earth using standard DNA barcodes and (ii) active development and application of extended DNA barcodes using genome skimming to augment the standard barcoding approach.     

Detection of invasive and cryptic species in marine mussels (Bivalvia,Mytilidae): A chromosomal perspective

Marine mussels illustrate a stunning variability in shape and color. Such variability, added to the scarcity of reliable morphological characters for their identification, can mislead recognition prompting the assignation of specimens of a single species to different ones or incorporate specimens belonging to different taxa into a single one. DNA barcoding is widely used for species identification; however, as this method relies on the previous morphological identification of the specimens, some of the DNA sequences stored in DNA databases are incorrectly assigned to a given species. In view of this uncertainty, further criteria beyond morphological characters and DNA sequences in databases are required to more reliably and accurately identify marine mussels. In this work we mapped ribosomal RNA and histone gene clusters to chromosomes of four species of marine mussels and compared them with those from another eight marine mussel taxa. Specimens of these twelve taxa were also DNA barcoded. Our results clearly demonstrated that the chromosomal analysis of marine mussels could shed light on their identification and, therefore, solve contradictions posed by morphological and molecular data.     

DNA barcoding reveals taxonomic conflicts in the Herichthys bartoni species group (Pisces: Cichlidae)

Fishes of the genus Herichthys are the only representatives of the family Cichlidae to have colonized the Neartic region. In this study, we used DNA barcode of 64 individuals of the Herichthys bartoni species group to test the monophyly of the species and the efficiency of this tool to discriminate among species. The Bayesian phylogenetic tree and the neighbour joining (NJ) tree obtained from the Kimura two‐parameter model (K2P) give similar topologies. DNA barcoding resolution was very poor (25%). Additionally, the low levels of genetic divergence among taxa preclude the use of threshold values as has been suggested in earlier studies.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

Long-term effects of local disturbance history on mobile stream invertebrates

The patchy distribution of benthic invertebrates in streams and rivers is an important and widely researched phenomenon. Previous studies on reasons for this patchiness have neglected the potential role of local disturbance history, probably because most lotic invertebrates are mobile and any effect of disturbance history was thought to be short-lived. Here we demonstrate for a New Zealand gravel-bed stream that local disturbance history can have long-term effects on the distribution of highly mobile stream invertebrates. Buried scour chains (100 at each of three 20-m sites within a 350-m reach) indicated that a spate with a return period of 5 months caused a mosaic of bed patches with different stabilities. More than 2 months after the spate, we took random, quantitative samples at each site from five patches that had experienced 4 cm or more of scour during the spate, from five patches with 4 cm or more of fill, and from five stable patches. Density of the dominant invertebrate taxon, the highly mobile mayfly Deleatidium spp., and densities of another three of the seven most common taxa differed significantly between patch stability categories. Larvae of Deleatidium, the black fly Austrosimulium spp. and the dipteran Eriopterini were most abundant in fill patches, whereas Isopoda were most abundant in scour patches. Total invertebrate densities and densities of six common taxa also differed between sites, although these were only 95–120 m apart. These results show that local disturbance history can have long-term effects on lotic invertebrates and be an important cause of invertebrate patchiness. The observed effects might have been even stronger had we sampled sooner after the spate or after a large flood. Disturbance history may influence invertebrates both directly (through dislodgement or mortality) and indirectly, through effects on the spatial distribution of their resources. Our results suggest that the role of disturbance in structuring animal communities dominated by mobile species may be more important than previously thought. Received: 25 January 2000 / Accepted: 14 April 2000     

Comprehensive DNA barcodes for species identification and discovery of cryptic diversity in mayfly larvae from South Korea: Implications for freshwater ecosystem biomonitoring

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing baseline reference for phylogenetic analysis and aquatic ecosystem biomonitoring. We obtained 112 novel sequences of the barcode region of the mitochondrial DNA cytochrome c oxidase subunit I gene representing 11 families, 25 genera, and 43 species of mayfly (Insecta: Ephemeroptera) from South Korea. No species shared barcode sequences and all can be identified with barcodes with a possible exception of some species. Minimum levels of interspecific genetic distances ranged from 6.7 to 32.9% (mean: 23.7%), whereas average levels of intraspecific divergence was 3.7%. The latter value was inflated by the presence of very high divergences within some taxa. In fact, approximately 33.3% (15/45) of the species included two or more haplotype clusters showing greater than 5.0% sequence divergence and some values were as high as 32.9%. Many of the species with high intraspecific divergences are para‐ or polyphyletic and represent the possibility of species complexes. Our study suggests that type or topotype specimens should be sequenced to identify accurate barcoding clusters with morphological species concepts and also to determine the status of currently synonymized species.     

DNA barcoding of fresh seafood in Australian markets reveals misleading labelling and sale of endangered species

Flake and shark samples were purchased from outlets in several coastal Australian regions and genetically barcoded using the cytochrome oxidase subunit 1 (CO1) gene to investigate labelling reliability and species-specific sources of ambiguously labelled fillets. Of the 41 shark fillet samples obtained, 23 yielded high-quality CO1 sequences, out of which 57% (n = 13) were labelled ambiguously (misleading) and 35% (n = 8) incorrectly. In contrast, barramundi fillets, which are widely available and sought after in Australian markets, were shown to be accurately labelled. Species identified from shark samples, including the shortfin mako (n = 3) and the scalloped hammerhead (n = 1), are assessed by the IUCN as endangered and critically endangered, respectively, with several others classified as vulnerable and near threatened.     

Scale-dependent effects of fine sediments on temperate headwater invertebrates

1. Anthropogenic activities can increase fine sediment supply to streams over multiple spatial and temporal extents. Identifying the processes responsible, and the scale at which any effects on stream organisms become evident, are key management needs, but appropriately scaled surveys are surprisingly few. 2. We surveyed macroinvertebrates and superficial fine sediments at two spatial resolutions (reach‐ and patch‐scale) in tributaries of the River Usk, a temperate, montane catchment in rural Wales (U.K.). Land use, habitat and geomorphological character were measured on‐site or derived from an existing database (=Fluvial Audit). We aimed to identify: (i) how in‐stream sediments varied with land use and associated geomorphology; (ii) likely consequences for macroinvertebrates and (iii) any scale‐dependence in relationships between macroinvertebrates and sediment character. 3. At both the reach‐ and patch‐scales, bed cover by fine sediment was related directly to the extent of eroding banks 500 m upstream. In turn, sedimentation and bank erosion were negatively correlated with catchment or riparian woodland extent. 4. At the reach scale, macroinvertebrate composition varied with catchment land use and stream chemistry, with richness declining as rough grazing or woodland was replaced by improved grassland. There was no response to deposited sediment except for weak increase in the relative abundance of oligochaetes. 5. By contrast, at the patch scale, fine sediments were accompanied by pronounced changes in invertebrate composition, and we ranked the 27 most common taxa according to their apparent sediment tolerance. General estimating equations showed that total and Ephemeroptera, Plecoptera and Trichoptera richness decreased significantly by 20% and 25% at the most sediment impacted sites (30% cover) by comparison with sediment‐free sites. 6. We conclude that sediment deposition in the upper Usk system mostly reflects local bank erosion, with riparian woodland likely to mediate this process through bank stability. Fine sediment release had marked ecological effects, but these were detectable only at patch‐to‐patch scales. We suggest that investigation of localized sediment release in streams will benefit from scale‐dependent or scale‐specific sampling, and some effects could go undetected unless sample resolution is selected carefully.     

Direct PCR of indigenous and invasive mosquito species: a time‐ and cost‐effective technique of mosquito barcoding

Millions of people die each year as a result of pathogens transmitted by mosquitoes. However, the morphological identification of mosquito species can be difficult even for experts. The identification of morphologically indistinguishable species, such as members of the Anopheles maculipennis complex (Diptera: Culicidae), and possible hybrids, such as Culex pipiens pipiens/Culex pipiens molestus (Diptera: Culicidae), presents a major problem. In addition, the detection and discrimination of newly introduced species can be challenging, particularly to researchers without previous experience. Because of their medical importance, the clear identification of all relevant mosquito species is essential. Using the direct polymerase chain reaction (PCR) method described here, DNA amplification without prior DNA extraction is possible and thus species identification after sequencing can be achieved. Different amounts of tissue (leg, head; larvae or adult) as well as different storage conditions (dry, ethanol, ?20 and ?80 °C) and storage times were successfully applied and showed positive results after amplification and gel electrophoresis. Overall, 28 different indigenous and non‐indigenous mosquito species were analysed using a gene fragment of the COX1 gene for species differentiation and identification by sequencing this 658‐bp fragment. Compared with standard PCR, this method is time‐ and cost‐effective and could thus improve existing surveillance and control programmes.     

High mitochondrial diversity in geographically widespread butterflies of Madagascar: A test of the DNA barcoding approach

The standardized use of mitochondrial cytochrome c oxidase subunit I (COI) gene sequences as DNA barcodes has been widely promoted as a high-throughput method for species identification and discovery. Species delimitation has been based on the following criteria: (1) monophyletic association and less frequently (2) a minimum 10× greater divergence between than within species. Divergence estimates, however, can be inflated if sister species pairs are not included and the geographic extent of variation within any given taxon is not sampled comprehensively. This paper addresses both potential biases in DNA divergence estimation by sampling range-wide variation in several morphologically distinct, endemic butterfly species in the genus Heteropsis, some of which are sister taxa. We also explored the extent to which mitochondrial DNA from the barcode region can be used to assess the effects of historical rainforest fragmentation by comparing genetic variation across Heteropsis populations with an unrelated forest-associated taxon Saribia tepahi. Unexpectedly, generalized primers led to the inadvertent amplification of the endosymbiont Wolbachia, undermining the use of universal primers and necessitating the design of genus-specific COI primers alongside a Wolbachia-specific PCR assay. Regardless of the high intra-specific genetic variation observed, most species satisfy DNA barcoding criteria and can be differentiated in the nuclear phylogeny. Nevertheless, two morphologically distinguishable candidate species fail to satisfy the barcoding 10× genetic distance criterion, underlining the difficulties of applying a standard distance threshold to species delimitation. Phylogeographic analysis of COI data suggests that forest fragmentation may have played an important role in the recent evolutionary diversification of these butterflies. Further work on other Malagasy taxa using both mitochondrial and nuclear data will provide better insight into the role of historical habitat fragmentation in species diversification and may potentially contribute to the identification of priority areas for conservation.     

Variable effects of sediment addition on stream benthos

Two upper Piedmont streams were studied to determine the effects of road construction, especially sediment inputs. Benthic macroinvertebrate data suggest that the stream community responded to sediment additions in two different ways. Under high flow conditions the benthic fauna occurs mainly on rocky substrates. As sediment is added to a stream the area of available rock habitat decreases, with a corresponding decrease in benthic density. There is, however, little change in community structure. Under low flow conditions, stable-sand areas may support high densities of certain taxa. Density of the benthic macroinvertebrates in these areas may be much greater than the density recorded in control areas, and there are distinct changes in community structure.     

DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH-psbA, trnL-F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear-cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty-seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH-psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH-psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.     

Genetic structure in a progenetic trematode: signs of cryptic species with contrasting reproductive strategies

Complexes of cryptic species are rapidly being discovered in many parasite taxa, including trematodes. However, after they are found, cryptic species are rarely distinguished from each other with respect to key ecological or life history traits. In this study, we applied an integrative taxonomic approach to the discovery of cryptic species within Stegodexamene anguillae, a facultatively progenetic trematode common throughout New Zealand. The presence of cryptic species was determined by the genetic divergence found in the mitochondrial cytochrome c oxidase I gene, the 16S rRNA gene and the nuclear 28S gene, warranting recognition of two distinct species and indicating a possible third species. Speciation was not associated with geographic distribution or microhabitat within the second intermediate host; however frequency of the progenetic reproductive strategy (and the truncated life cycle associated with it) was significantly greater in one of the lineages. Therefore, two lines of evidence, molecular and ecological, support the distinction between these two species and suggest scenarios for their divergence.