Preliminary phylogeny of Encarsia Förster (Hymenoptera: Aphelinidae) based on morphology and 28S rDNA

Species of Encarsia F?rster (Hymenoptera: Aphelinidae, Coccophaginae) are economically important for the biological control of whitefly and armored scale pests (Hemiptera: Aleyrodidae, Diaspididae). Whereas some regional keys for identification of Encarsia species are now available, few studies have addressed relationships within this diverse and cosmopolitan genus because of unreliable morphological data. Nuclear sequences of the D2 expansion region of 28S rDNA were determined from 67 strains of 24 species representing 10 species groups of Encarsia, 2 strains of Encarsiella noyesi Hayat, and 1 strain of Coccophagoides fuscipennis Girault. Analysis of molecular data alone and combined with morphological data resolves many nodes not resolved by morphology alone and offer insights into which morphological characters are useful for supporting group relationships. All analyses that include molecular data reveal Encarsia to be paraphyletic with respect to Encarsiella. If monophyly of Encarsia is constrained, the relationships are the same but with a different root within Encarsia, and these trees are presented as an alternate hypothesis. The luteola and strenua species groups are shown by both morphological and molecular data to be monophyletic, whereas the inaron group, the E. nigricephala + luteola group, and the E. quericola + strenua group are supported only by molecular data. The aurantii and parvella species groups are not supported in any of the analyses. The utility of morphological characters for defining species group relationships is discussed.     

Barcoding of biting midges in the genus Culicoides: a tool for species determination

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.     

A combination of molecular and morphological approaches resolves species in the taxonomically difficult genus Procladius Skuse (Diptera: Chironomidae) despite high intra-specific morphological variation

Molecular approaches for identifying aquatic macroinvertebrate species are increasingly being used but there is ongoing debate about the number of DNA markers needed to differentiate species accurately. Here, we use two mitochondrial genes (cytochrome oxidase I, cytochrome b) and a nuclear gene (carbamoylphosphate synthetase) to differentiate species variation within the taxonomically challenging chironomid genus Procladius from southern Australia, a genus which is important for pollution monitoring. The mitochondrial genes indicated cryptic species that were subsequently linked to morphological variation at the larval and pupal stage. Two species previously described based on morphological criteria were linked to molecular markers, and there was evidence for additional cryptic species. Each genetic marker provided different information, highlighting the importance of considering multiple genes when dissecting taxonomically difficult groups, particularly those used in pollution monitoring.     

Reproductive isolation among cryptic species in the ectomycorrhizal genus Strobilomyces: population-level CAPS marker-based genetic analysis

Among the higher fungi, reproductively isolated cryptic species exist that are morphologically difficult to distinguish owing to a lack of taxonomically useful morphological characters. Mating tests are helpful for detecting reproductive isolation between cryptic species, but are often difficult to perform for higher fungi, especially ectomycorrhizal fungi. In order to identify cryptic species of the ectomycorrhizal genus Strobilomyces more efficiently, lineages were defined based on the nucleotide sequence of two mitochondrial genes. Then the gene flow among lineages was measured using cleaved amplified polymorphic sequences (CAPS) markers designed for single copy nuclear genes. No heterozygosity was observed between different lineages, but within the same lineage heterozygosity was present at the ratio expected given Hardy Weinberg equilibrium. These results show that the mtDNA lineages are separate Mendelian populations, possibly cryptic species that are reproductively isolated from each other.     

Revision of the systematics of the genus Calliptamus Serville 1831, (Orthoptera: Acrididae: Calliptaminae) in Algeria using morphological,chemical, and genetic data

Summary. The genus Calliptamus contains swarming orthopterans that cause serious damage in Algerian agricultural systems. However, it remains difficult to identify species within this genus; a thorough understanding of the group’s systematics and the utilization of novel taxonomic criteria are needed. We used morphological analysis along with two other methods of species identification – chemotaxonomy with cuticular compounds and DNA barcoding involving the COI gene – to classify 81 individual grasshoppers collected at two different sites in the Sétif region (northeastern Algeria). The chemotaxonomic analyses yielded ambiguous results, but DNA barcoding allowed us to differentiate two Calliptamus species found in Algeria: Calliptamus barbarus (Costa 1836), and Calliptamus wattenwylianus (Pantel 1896). Several morphological criteria used in identification keys appear to reflect differences among morphotypes rather than differences between species, and their taxonomic specificity is not supported by the barcoding data. The number of spines on the hind tibia is the only morphological criterion that reflected genetic differences between species; it is thus considered to be a taxonomically useful feature for identifying species in this genus.     

Use of nuclear and mitochondrial DNA PCR and sequencing for molecular identification of Diphyllobothrium isolates potentially infective for humans

Tapeworms of the genus Diphyllobothrium (Cobold, 1858) are widely distributed all around the world and some of them are agents of human diphyllobothriasis. Approximately 50 species have been described within the Diphyllobothrium genus but only 13 are human pathogens. Species identification by using morphological criteria is very difficult. We determined the value of 18S ribosomal RNA gene, internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 gene (COI) sequences to differentiate between Diphyllobothrium isolates. Sequences from 18 isolates (larvae or adults) of D. latum, D. nihonkaiense, D. ditremum, D. dentriticum and D. stemmacephalum species were obtained. COI region sequences analysis was clearly more discriminative than those of the ITS1 and 18S rRNA and was a useful tool for identifying specimens.     

Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free‐ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post‐mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already‐described taeniid species. Indeed, we detected three well‐defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.     

COI barcodes for identification of Merodon hoverflies (Diptera, Syrphidae) of Lesvos Island, Greece

DNA barcoding has become a useful system for linking different biological life stages, and for identification of species within a known taxonomic framework. In this study, we generated mitochondrial DNA COI barcodes using adult specimens of all 22 species of the hoverfly genus Merodon (Diptera, Syrphidae) occurring on Lesvos island (Greece). The generated COI barcodes could well discriminate between all Merodon taxa of Lesvos, except for M. loewi and M. papillus that shared the same haplotype, despite their clear morphological differences. In addition, the barcodes revealed two cases of hitherto unknown morphologically cryptic species close to M. avidus and M. nigritarsis, respectively. Because only few successful rearings of immature stages of Merodon hoverflies are available, the larval host plant remains unknown for these phytophagous taxa. The obtained COI barcode library for the Merodon spp. of Lesvos will constitute a tool to link any unknown immature stages with already known species, and thus provide important life-history information and promise for ecological studies.     

Diversity of the Southeast Asian leaf turtle genus Cyclemys: how many leaves on its tree of life?

In the present study, we use mtDNA sequence data (cyt b gene) in combination with nuclear DNA sequences (C-mos, Rag2 genes, R35 intron), nuclear genomic fingerprints (ISSR) and morphological data to reveal species diversity within the Southeast Asian leaf turtle genus Cyclemys , a morphologically difficult group comprising cryptic species. Two morphologically distinct major groupings exist, a yellow-bellied species group with three taxa ( Cyclemys atripons , C. dentata , C. pulchristriata ) and a dark-bellied species group. The latter contains besides the morphologically variable C. oldhamii three additional new species ( C. enigmatica n. sp., C. fusca n. sp., C. gemeli n. sp.). According to mtDNA data, C. fusca and C. gemeli constitute with high support the sister group of a clade comprising all other species, indicating that the dark-bellied species are not monophyletic, despite morphological similarity. mtDNA sequences of C. enigmatica , being highly distinct in nuclear genomic markers, do not differ from the sympatric C. dentata , suggesting that the original mitochondrial genome of C. enigmatica was lost due to introgressive hybridization. Morphological discrimination of Cyclemys species is possible using multivariate methods. However, gross morphology of most dark-bellied species on the one hand and of C. atripons and C. pulchristriata on the other is so similar that reliable species determination is only possible when genetic markers are used. The high diversity within Cyclemys requires revision of the IUCN Red List Categories for leaf turtles because the former assessment was based on the wrong assumption that in the entire range of the genus occurs only a single species.     

DNA Barcoding for the Identification of Sand Fly Species (Diptera,Psychodidae, Phlebotominae) in Colombia

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia.     

Genetic barcoding of commercial Bêche-de-mer species (Echinodermata: Holothuroidea)

There are more than 47 species of holothurians used for bêche-de-mer production, many of which are locally overfished. With three exceptions, all bêche-de-mer species are Aspidochirotida and species identification of many of these is difficult. We analysed available genetic information and newly generated sequences to determine if genetic barcoding with the mitochondrial COI gene can be used to identify bêche-de-mer species. Although genetic data were available for ～50% of bêche-de-mer species, sufficient information and within-species replication were only available for six species. We generated 96 new COI sequences extending the existing database to cover most common species. COI unambiguously identified most bêche-de-mer species providing a genetic barcode for the identification of known species. In addition, conspecific (1.3%) variation and congeneric (16.9%) divergence were well separated ('barcoding-gap') albeit with a small overlap, which may lead to some error if genetic sampling alone was applied for species discovery. In addition to identification of adults, COI sequences were useful to identify juveniles that are often morphologically different. Sequence data showed that large (deep) and small (shallow) morphotypes of Holothuria atra are the same species, but suggested potential cryptic species within this taxon. For bêche-de-mer, the COI barcode proved useful in species clarification and discovery, but further genetic and taxonomic work is essential for several species. Some bêche-de-mer clades were problematic with morphologically disparate specimens sharing the same barcode. Our study indicated the presence of undescribed species (Bohadschia sp.) and species that constitute separate species in the Indian and Pacific Ocean (e.g. Holothuria fuscogilva).     

Molecular Barcoding of Aquatic Oligochaetes: Implications for Biomonitoring

Aquatic oligochaetes are well recognized bioindicators of quality of sediments and water in watercourses and lakes. However, the difficult taxonomic determination based on morphological features compromises their more common use in eco-diagnostic analyses. To overcome this limitation, we investigated molecular barcodes as identification tool for broad range of taxa of aquatic oligochaetes. We report 185 COI and 52 ITS2 rDNA sequences for specimens collected in Switzerland and belonging to the families Naididae, Lumbriculidae, Enchytraeidae and Lumbricidae. Phylogenetic analyses allowed distinguishing 41 lineages separated by more than 10 % divergence in COI sequences. The lineage distinction was confirmed by Automatic Barcode Gap Discovery (ABGD) method and by ITS2 data. Our results showed that morphological identification underestimates the oligochaete diversity. Only 26 of the lineages could be assigned to morphospecies, of which seven were sequenced for the first time. Several cryptic species were detected within common morphospecies. Many juvenile specimens that could not be assigned morphologically have found their home after genetic analysis. Our study showed that COI barcodes performed very well as species identifiers in aquatic oligochaetes. Their easy amplification and good taxonomic resolution might help promoting aquatic oligochaetes as bioindicators for next generation environmental DNA biomonitoring of aquatic ecosystems.     

Barcoding bugs: DNA-based identification of the true bugs (Insecta: Hemiptera: Heteroptera)

Background

DNA barcoding, the analysis of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene, has been shown to provide an efficient method for the identification of species in a wide range of animal taxa. In order to assess the effectiveness of barcodes in the discrimination of Heteroptera, we examined 344 species belonging to 178 genera, drawn from specimens in the Canadian National Collection of Insects.

Methodology/Principal Findings

Analysis of the COI gene revealed less than 2% intra-specific divergence in 90% of the taxa examined, while minimum interspecific distances exceeded 3% in 77% of congeneric species pairs. Instances where barcodes fail to distinguish species represented clusters of morphologically similar species, except one case of barcode identity between species in different genera. Several instances of deep intraspecific divergence were detected suggesting possible cryptic species.

Conclusions/Significance

Although this analysis encompasses 0.8% of the described global fauna, our results indicate that DNA barcodes will aid the identification of Heteroptera. This advance will be useful in pest management, regulatory and environmental applications and will also reveal species that require further taxonomic research.     

Patterns of speciation inferred from mitochondrial DNA in North American Chthamalus (Cirripedia: Balanomorpha: Chthamaloidea)

Chthamalus is a cosmopolitan genus of high intertidal barnacles that are difficult to distinguish morphologically. This study focuses on a single subgeneric group of Chthamalus that is found on the coasts of North and Central America to determine the age and pattern of speciation among these species. Two comparisons of genetic divergence are made across the Panamanian Isthmus, allowing estimates of the substitution rate to be made for two mitochondrial genes coding for COI and 16S rRNA in these barnacles. These data suggest that the little morphological diversification that there has been in Chthamalus occurred early in the history of the genus, and subsequent radiations were probably induced by transient periods of population separation during late Miocene to Recent climatic changes.     

Large amounts of genetic divergence among Italian species of the genus Orchesella (Insecta, collembola) and the relationships of two new species

The phylogenetic position of two putative new species of the collembolan genus Orchesella was investigated by comparison with four other Italian species of the genus using a fragment of the mitochondrial gene encoding for subunit I of cytochrome c oxidase (COI). The gene showed the well-known A + T bias, typical of insect mitochondrial DNA, although A + T content was not as high as that observed in species belonging to more derived insect orders. The large number of variable sites in 3rd codon positions (85.2% variable) suggested that these sites contain significant homoplasy due to multiple hits. Despite the lack of morphological differentiation, the COI portion examined shows remarkable levels of genetic divergence between the putative species and their closest relatives. Phylogenetic analysis suggests that one of the putative new species is related to O. villosa, whereas the other is included in a clade with O. cincta and O. ranzii. The species O. chiantica appears to be related to O. villosa, agreeing with previous allozyme data.     

DNA barcoding of endoparasitoid wasps in the genus Anicetus reveals high levels of host specificity (Hymenoptera: Encyrtidae)

The genus Anicetus includes economically important biocontrol agents that are introduced for control of soft and wax scale insect agricultural pests (Ceroplastes spp.). Understanding of host–parasitoid associations is critical to the successful outcome of their utilization in biological control projects. However, identification of these parasitoids is often difficult because of their small size and generally similar morphological features, and hence, studies on the host–parasitoid associations. Here, nucleotide sequence data were generated from the mitochondrial COI gene and the D2 region of 28S rRNA to assess genetic variation within and between species of Anicetus occurring in China. The results of this study support the use of the COI and the D2 region of 28S rRNA gene as useful markers in separating species of Anicetus, even in cases where morphological differences are subtle. On the other hand, the COI gene is also useful in recognizing species with much variation in morphology. DNA barcoding reveals high levels of host specificity of endoparasitoids wasps in the genus Anicetus. Our results indicate that each Anicetus species is adapted to a limited set of host species, or even are monospecific in their host choice.     

Cryptic diversity in Brevipalpus mites (Tenuipalpidae)

Defining the taxonomic identity of organisms is a prerequisite for their study, and in the case of economically important species, misidentification may lead to the application of inappropriate prevention and control strategies. Flat mites of the Brevipalpus genus include several crop pests and the systematics of these mites represents a challenge for acarologists. Many of the most economically important Brevipalpus species have repeatedly been inaccurately identified. Such problematic classification has been attributed to the likely occurrence of cryptic species in the genus. In this study, we used an integrative approach that combined molecular analyses, including sequence‐based species delimitation, with detailed morphological identification using traits that have recently showed to be taxonomically informative. Sequences of mitochondrial cytochrome c oxidase subunit I (COI) were obtained from individuals collected from host plants belonging to 14 genera and 13 families across 29 locations in the Americas (Brazil, Chile, USA). The phylogenetic analyses included previously published Brevipalpus sequences from GenBank, and the final data set was classified into 44 haplotypes. Six putative species were recognised by COI‐based species delimitation analysis, and morphological evidence supported each of these species. The integrative approach revealed the occurrence of cryptic species in the Brevipalpus genus and contributed to the clarification of previously noted incongruences. The results presented here allow for the evaluation of taxonomic characteristics in a phylogenetic context and indicate new characters for the differentiation of Brevipalpus species. In addition, Brevipalpus incognitus n. sp. Ferragut & Navia, a cryptic species detected in this study, is described based on morphological and molecular traits. Implications of the advances in Brevipalpus systematics presented herein with respect to pest management are briefly discussed.     

DNA Barcoding to Improve the Taxonomy of the Afrotropical Hoverflies (Insecta: Diptera: Syrphidae)

The identification of Afrotropical hoverflies is very difficult because of limited recent taxonomic revisions and the lack of comprehensive identification keys. In order to assist in their identification, and to improve the taxonomy of this group, we constructed a reference dataset of 513 COI barcodes of 90 of the more common nominal species from Ghana, Togo, Benin and Nigeria (W Africa) and added ten publically available COI barcodes from nine nominal Afrotropical species to this (total: 523 COI barcodes; 98 nominal species; 26 genera). The identification accuracy of this dataset was evaluated with three methods (K2P distance-based, Neighbor-Joining (NJ) / Maximum Likelihood (ML) analysis, and using SpeciesIdentifier). Results of the three methods were highly congruent and showed a high identification success. Nine species pairs showed a low (< 0.03) mean interspecific K2P distance that resulted in several incorrect identifications. A high (> 0.03) maximum intraspecific K2P distance was observed in eight species and barcodes of these species not always formed single clusters in the NJ / ML analayses which may indicate the occurrence of cryptic species. Optimal K2P thresholds to differentiate intra- from interspecific K2P divergence were highly different among the three subfamilies (Eristalinae: 0.037, Syrphinae: 0.06, Microdontinae: 0.007–0.02), and among the different general suggesting that optimal thresholds are better defined at the genus level. In addition to providing an alternative identification tool, our study indicates that DNA barcoding improves the taxonomy of Afrotropical hoverflies by selecting (groups of) taxa that deserve further taxonomic study, and by attributing the unknown sex to species for which only one of the sexes is known.     

COI barcoding of true bugs (Insecta, Heteroptera)

Several recent studies have proposed that partial DNA sequences of the cytochrome c oxidase I (COI) mitochondrial gene might serve as DNA barcodes for identifying and differentiating between animal species, such as birds, fish and insects. In this study, we tested the effectiveness of a COI barcode to identify true bugs from 139 species collected from Korea and adjacent regions (Japan, Northeastern China and Fareast Russia). All the species had a unique COI barcode sequence except for the genus Apolygus (Miridae), and the average interspecific genetic distance between closely related species was about 16 times higher than the average intraspecific genetic distance. DNA barcoding identified one probable new species of true bug and revealed identical or very recently divergent species that were clearly distinguished by morphological characteristics. Therefore, our results suggest that COI barcodes can reveal new cryptic true bug species and are able to contribute for the exact identification of the true bugs.