Molecular Identification of Birds: Performance of Distance-Based DNA Barcoding in Three Genes to Delimit Parapatric Species

Background

DNA barcoding based on the mitochondrial cytochrome oxidase subunit I gene (cox1 or COI) has been successful in species identification across a wide array of taxa but in some cases failed to delimit the species boundaries of closely allied allopatric species or of hybridising sister species.

Methodology/Principal Findings

In this study we extend the sample size of prior studies in birds for cox1 (2776 sequences, 756 species) and target especially species that are known to occur parapatrically, and/or are known to hybridise, on a Holarctic scale. In order to obtain a larger set of taxa (altogether 2719 species), we include also DNA sequences of two other mitochondrial genes: cytochrome b (cob) (4614 sequences, 2087 species) and 16S (708 sequences, 498 species). Our results confirm the existence of a wide gap between intra- and interspecies divergences for both cox1 and cob, and indicate that distance-based DNA barcoding provides sufficient information to identify and delineate bird species in 98% of all possible pairwise comparisons. This DNA barcoding gap was not statistically influenced by the number of individuals sequenced per species. However, most of the hybridising parapatric species pairs have average divergences intermediate between intraspecific and interspecific distances for both cox1 and cob.

Conclusions/Significance

DNA barcoding, if used as a tool for species discovery, would thus fail to identify hybridising parapatric species pairs. However, most of them can probably still assigned to known species by character-based approaches, although development of complementary nuclear markers will be necessary to account for mitochondrial introgression in hybridising species.     

High-Level Diversity of Dinoflagellates in the Natural Environment,Revealed by Assessment of Mitochondrial cox1 and cob Genes for Dinoflagellate DNA Barcoding

DNA barcoding is a diagnostic technique for species identification using a short, standardized DNA. An effective DNA barcoding marker would be very helpful for unraveling the poorly understood species diversity of dinoflagellates in the natural environment. In this study, the potential utility for DNA barcoding of mitochondrial cytochrome c oxidase 1 (cox1) and cytochrome b (cob) was assessed. Among several primer sets examined, the one amplifying a 385-bp cob fragment was most effective for dinoflagellates. This short cob fragment is easy to sequence and yet possess reasonable taxon resolution. While the lack of a uniform gap between interspecific and intraspecific distances poses difficulties in establishing a phylum-wide species-discriminating distance threshold, the variability of cob allows recognition of species within particular lineages. The potential of this cob fragment as a dinoflagellate species marker was further tested by applying it to an analysis of the dinoflagellate assemblages in Long Island Sound (LIS) and Mirror Lake in Connecticut. In LIS, a highly diverse assemblage of dinoflagellates was detected. Some taxa can be identified to the species and some to the genus level, including a taxon distinctly related to the bipolar species Polarella glacialis, and the large number of others cannot be clearly identified, due to the inadequate database. In Mirror Lake, a Ceratium species and an unresolved taxon were detected, exhibiting a temporal transition from one to the other. We demonstrate that this 385-bp cob fragment is promising for lineage-wise dinoflagellate species identification, given an adequate database.DNA barcoding is a diagnostic technique for species identification using a short, standardized DNA (i.e., DNA barcode) (15). For microbial organisms, this PCR-based technique is useful not only for identifying cultured species but also for rapid retrieval and species identification for uncultured taxa from natural environments. A good DNA barcoding marker should be simple (easy to PCR amplify and sequence) and universal (effective for a wide range of lineages), with a high resolving power (high interspecific and low intraspecific variations). Therefore, an ideal DNA barcoding marker is a relatively short and reasonably variable gene fragment (for species discrimination) flanked by highly conserved sequences (for primer design). The pioneering DNA barcoding work used mitochondrial cytochrome c oxidase 1 (cox1) to identify animal species (9, 10). Mitochondrial genes are a good barcode choice for animals, because they are markedly more variable than nuclear genes (3, 32) and contain conserved regions for primer design. Among other organisms, cox1 has also been shown to be useful for barcoding other organisms, such as fungi (35). Initial attempts at cox1 barcoding for macroalgae (rhodophyte and phaeophyte) also showed good potential (21, 29, 34). In land plants, the mitochondrial genome evolves substantially more slowly than the nuclear genome (26, 27), rendering its genes less useful than genes from chloroplast (14, 15). The utility of cox1 or other mitochondrial genes for DNA barcoding is less clear for unicellular organisms, with few documented attempts (e.g., reference 7) for those living in the marine ecosystem.Dinoflagellates are important unicellular organisms in the marine ecosystem because of their significant contribution to marine primary production, support of coral reef growth through symbiotic associations (31), micrograzing (25), and formation of harmful and often toxic algal blooms (1). Dinoflagellates are genetically diverse, with at least 2,000 documented extant species and 2,000 fossil species. Continual discovery of new species in the ocean (e.g., references 6, 12, 13, 17, 20, 22, 24, and 38) suggests that there are likely many more dinoflagellate lineages to be recognized. Identification of dinoflagellate species and discovery of species diversity by use of traditional morphological analysis is often hampered by high degrees of morphological similarity and a lack of unique characters between different species. A systematic survey of dinoflagellate diversity using a diagnostic molecular marker is highly desirable. To unravel species diversity and new taxa in natural environments, a DNA barcode would need to be specific for dinoflagellates in addition to the above-mentioned requirements.In this study, the potential utilities of mitochondrial genes as DNA barcoding markers were assessed. Mitochondrial cox1 and cob (the gene coding for cytochrome b) from dinoflagellates were compared for PCR efficiency and resolving power. We demonstrated that while neither of the mitochondrial genes seems to be a good phylum-wide DNA barcoding marker, a cob primer set can be used to determine the species diversity of dinoflagellate flora in a lineage-by-lineage manner.     

Deciphering amphibian diversity through DNA barcoding: chances and challenges

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.     

Does the DNA barcoding gap exist? – a case study in blue butterflies (Lepidoptera: Lycaenidae)

Background

DNA barcoding, i.e. the use of a 648 bp section of the mitochondrial gene cytochrome c oxidase I, has recently been promoted as useful for the rapid identification and discovery of species. Its success is dependent either on the strength of the claim that interspecific variation exceeds intraspecific variation by one order of magnitude, thus establishing a "barcoding gap", or on the reciprocal monophyly of species.

Results

We present an analysis of intra- and interspecific variation in the butterfly family Lycaenidae which includes a well-sampled clade (genus Agrodiaetus) with a peculiar characteristic: most of its members are karyologically differentiated from each other which facilitates the recognition of species as reproductively isolated units even in allopatric populations. The analysis shows that there is an 18% overlap in the range of intra- and interspecific COI sequence divergence due to low interspecific divergence between many closely related species. In a Neighbour-Joining tree profile approach which does not depend on a barcoding gap, but on comprehensive sampling of taxa and the reciprocal monophyly of species, at least 16% of specimens with conspecific sequences in the profile were misidentified. This is due to paraphyly or polyphyly of conspecific DNA sequences probably caused by incomplete lineage sorting.

Conclusion

Our results indicate that the "barcoding gap" is an artifact of insufficient sampling across taxa. Although DNA barcodes can help to identify and distinguish species, we advocate using them in combination with other data, since otherwise there would be a high probability that sequences are misidentified. Although high differences in DNA sequences can help to identify cryptic species, a high percentage of well-differentiated species has similar or even identical COI sequences and would be overlooked in an isolated DNA barcoding approach.     

Assessing DNA Barcoding as a Tool for Species Identification and Data Quality Control

In recent years, the number of sequences of diverse species submitted to GenBank has grown explosively and not infrequently the data contain errors. This problem is extensively recognized but not for invalid or incorrectly identified species, sample mixed-up, and contamination. DNA barcoding is a powerful tool for identifying and confirming species and one very important application involves forensics. In this study, we use DNA barcoding to detect erroneous sequences in GenBank by evaluating deep intraspecific and shallow interspecific divergences to discover possible taxonomic problems and other sources of error. We use the mitochondrial DNA gene encoding cytochrome b (Cytb) from turtles to test the utility of barcoding for pinpointing potential errors. This gene is widely used in phylogenetic studies of the speciose group. Intraspecific variation is usually less than 2.0% and in most cases it is less than 1.0%. In comparison, most species differ by more than 10.0% in our dataset. Overlapping intra- and interspecific percentages of variation mainly involve problematic identifications of species and outdated taxonomies. Further, we detect identical problems in Cytb from Insectivora and Chiroptera. Upon applying this strategy to 47,524 mammalian CoxI sequences, we resolve a suite of potentially problematic sequences. Our study reveals that erroneous sequences are not rare in GenBank and that the DNA barcoding can serve to confirm sequencing accuracy and discover problems such as misidentified species, inaccurate taxonomies, contamination, and potential errors in sequencing.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

Are the mitochondrial cox1 and cob genes suitable markers for species of Dinophysis Ehrenberg?

The identification of Dinophysis species with similar morphology but different toxic (Diarrhetic Shellfish Poisoning, DSP) potential is a crucial task in harmful algae monitoring programmes. The taxonomic assignment of Dinophysis species using molecular markers is a difficult task due to extremely low interspecific variability within their nuclear ribosomal genes and intergenic regions. Mitochondrial cox1 gene has been proposed as a better specific marker for Dinophysis species based on its higher resolution for two morphologically related species (Dinophysis acuminata and Dinophysis ovum) of the “Dinophysis acuminata complex”. In this study, the potential of two mitochondrial genes (mt cox1 and cob) to discriminate among six Dinophysis species (field isolates and cultures) associated with DSP events was explored. Neither mt cox1 nor cob genes provided enough resolution for all species of Dinophysis. The cob gene showed very poor resolution and grouped all Dinophysis spp. in a common clade. In contrast, the cox1 phylogeny distinguished 5 clades in the Dinophysiales – the “acuminata complex”, the “caudata group”, “acuta + norvegica” and Phalacromaspp. However, within the “D. acuminata complex” mtcox1 is so far the unique marker that differentiates D. acuminata from other species: isolates of D. ovum and Dinophysis sacculus had almost identical sequences (only four mismatches), but they were well separated from D. acuminata. D. acuminata and Dinophysis skagii (considered a life cycle stage of the former) showed identical cox1 sequences. Probes towards this gene can be useful in Mediterranean and Western Iberia sites where the co-occurrence of close morphotypes of D. acuminata and D. sacculus pose a problem for monitoring analyses. This is the first report on cultures of D. sacculus and its phylogenetic relation with other species of the D. acuminata complex.     

A new species of the genus <Emphasis Type="Italic">Morellia</Emphasis> Robineau-Desvoidy (Diptera: Muscidae) from Yunnan,China, with analysis of available DNA barcoding sequences

A new species of the genus Morellia Robineau-Desvoidy, 1830, Morellia (Morellia) trifurcata sp. n., collected from Yunnan, China is described. Four DNA sequences of the partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene of this new species are provided. In order to evaluate the availability of DNA barcoding for identifying Morellia species, 38 currently available, non-identical COI sequences of 16 Morellia species are involved in a molecular analysis using the neighbor-joining (NJ) method. The intra- and interspecific p-distances are summarized.     

DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNA sequences

An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL–F, and psbA–trnH from the chloroplast genome, and ITS, ITS1, and ITS2 from the nuclear genome) and three combinations (rbcL + matK, psbA–trnH + ITS1, and trnL–F + ITS1). The present study collected 232 sequences from 27 Salvia species through DNA sequencing and 77 sequences within the same taxa from the GenBank. The discriminatory capabilities of these regions were evaluated in terms of PCR amplification success, intraspecific and interspecific divergence, DNA barcoding gaps, and identification efficiency via a tree-based method. ITS1 was superior to the other marker for discriminating between species, with an accuracy of 81.48%. The three combinations did not increase species discrimination. Finally, we found that ITS1 is a powerful barcode for identifying Salvia species, especially Salvia miltiorrhiza.     

A Three-Gene Dinoflagellate Phylogeny Suggests Monophyly of Prorocentrales and a Basal Position for <Emphasis Type="Italic">Amphidinium</Emphasis> and <Emphasis Type="Italic">Heterocapsa</Emphasis>

Many outstanding questions about dinoflagellate evolution can potentially be resolved by establishing a robust phylogeny. To do this, we generated a data set of mitochondrial cytochrome b (cob) and mitochondrial cytochrome c oxidase 1 (cox1) from a broad range of dinoflagellates. Maximum likelihood, maximum parsimony, and Bayesian methods were used to infer phylogenies from these genes separately and as a concatenated alignment with and without small subunit (SSU) rDNA sequences. These trees were largely congruent in topology with previously published phylogenies but revealed several unexpected results. Prorocentrum benthic and planktonic species previously placed in different clusters formed a monophyletic group in all trees, suggesting that the Prorocentrales is a monophyletic group. More strikingly, our analyses placed Amphidinium and Heterocapsa as early splits among dinoflagellates that diverged after the emergence of O. marina. This affiliation received strong bootstrap support, but these lineages exhibited relatively long branches. The approximately unbiased (AU-) test was used to assess this result using a three-gene (cob + cox1 + SSU rDNA) DNA data set and the inferred tree. This analysis showed that forcing Amphidinium or Heterocapsa to relatively more derived positions in the phylogeny resulted in significantly lower likelihood scores, consistent with the phylogenies. The position of these lineages needs to be further verified. Reviewing Editor: Dr. Martin Kreitman     

Description of a new species of Moniliformis (Acanthocephala: Moniliformidae) from Peromyscus hylocetes (Rodentia: Cricetidae) in Mexico

Moniliformis ibunami n. sp., is described from the intestine of the transvolcanic deermouse Peromyscus hylocetes Merriam 1898 (Cricetidae) from Parque Nacional Nevado de Colima “El Floripondio”, Jalisco, Mexico. The new species can be distinguished morphologically from the other 18 congeneric species of Moniliformis by a combination of morphological and molecular characters including the number of hooks on the proboscis (12 longitudinal rows, each one with six to eight transversally arranged unrooted hooks), the proboscis length (230–270 μm), the female trunk length (159–186 mm) and egg size (40–70 × 20–40). For molecular distinction, nearly complete sequences of the small subunit (SSU) and large subunit (LSU) of the nuclear ribosomal DNA and cytochrome oxidase subunit 1 (cox 1) of the mitochondrial DNA of the new species were obtained and compared with available sequences downloaded from GenBank. Phylogenetic analyses inferred with the three molecular markers consistently showed that Moniliformis ibunami n. sp. is sister to other congeneric species of Moniliformis. The genetic distance with cox 1 gene among Moniliformis ibunami n. sp., M. saudi, M. cryptosaudi, M. kalahariensis, M. necromysi and M. moniliformis ranged from 20 to 27%. Morphological evidence and high genetic distance, plus the phylogenetic analyses, indicate that acanthocephalans collected from the intestines of transvolcanic deer mice represent a new species which constitutes the seventh species of the genus Moniliformis in the Americas.     

Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

Background

DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses.

Methodology

We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs.

Results

The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183.

Conclusions

The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.     

The complete mitochondrial genome of the verongid sponge Aplysina cauliformis: implications for DNA barcoding in demosponges

DNA “barcoding,” the determination of taxon-specific genetic variation typically within a fragment of the mitochondrial cytochrome oxidase 1 (cox1) gene, has emerged as a useful complement to morphological studies, and is routinely used by expert taxonomists to identify cryptic species and by non-experts to better identify samples collected during field surveys. The rate of molecular evolution in the mitochondrial genomes (mtDNA) of nonbilaterian animals (sponges, cnidarians, and placozoans) is much slower than in bilaterian animals for which DNA barcoding strategies were developed. If sequence divergence among nonbilaterian mtDNA and specifically cox1 is too slow to generate diagnostic variation, alternative genes for DNA barcoding and species-level phylogenies should be considered. Previous study across the Aplysinidae (Demospongiae, Verongida) family of sponges demonstrated no nucleotide substitutions in the traditional cox1 barcoding fragment among the Caribbean species of Aplysina. As the mitochondrial genome of Aplysina fulva has previously been sequenced, we are now able to make the first comparisons between complete mtDNA of congeneric demosponges to assess whether potentially informative variation exists in genes other than cox1. In this article, we present the complete mitochondrial genome of Aplysina cauliformis, a circular molecule 19620 bp in size. The mitochondrial genome of A. cauliformis is the same length as is A. fulva and shows six confirmed nucleotide differences and an additional 11 potential SNPs. Of the six confirmed SNPs, NADH dehydrogenase subunit 5 (nad5) and nad2 each contain two, and in nad2 both yield amino acid substitutions, suggesting balancing selection may act on this gene. Thus, while the low nucleotide diversity in Caribbean aplysinid cox1 extends to the entire mitochondrial genome, some genes do display variation. If these represent interspecific differences, then they may be useful alternative markers for studies in recently diverged sponge clades.     

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome

Summary In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain ana ^r -14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome - c oxidase - cob gene encoding apocytochrome b - cox1I1, cox1I2a, cox1I2b, cox1I3 introns in cox1 - cox1Ix ^+/– indicates the presence or absence of the intron either in the native gene or after intron DNA excision - cox1Ix is a deletion in the intron leading to respiratory deficiency     

Identification of phlebotomine sand flies (Diptera: Psychodidae) from leishmaniasis endemic areas in southeastern Mexico using DNA barcoding

Leishmaniasis, a vector‐borne disease transmitted to humans through the bite of phlebotomine sand flies, is of public health significance in southeastern Mexico. Active and continuous monitoring of vectors is an important aspect of disease control for the prediction of potential outbreaks. Thus, the correct identification of vectors is paramount in this regard. In this study, we employed DNA barcoding as a tool for identifying phlebotomine sand flies collected in localized cutaneous leishmaniasis endemic areas of Quintana Roo, Mexico. Specimens were collected using CDC light and Shannon traps as part of the Mexican Ministry of Health surveillance program. DNA extraction was carried out using a nondestructive protocol, and morphological identification based on taxonomic keys was conducted on slide‐mounted specimens. Molecular taxonomic resolution using the 658‐bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was 100% congruent with the morphological identification. Seven species were identified: Lutzomyia cruciata (Coquillett 1907), Lutzomyia longipalpis (Lutz & Neiva 1912), Psathyromyia shannoni (Dyar 1929), Dampfomyia deleoni (Fairchild & Hertig 1947), Dampfomyia beltrani/steatopyga (Vargas & Díaz‐Nájera 1951), Bichromomyia olmeca olmeca (Vargas & Díaz‐Nájera, 1959), and Brumptomyia mesai (Sherlock 1962). Mean intraspecific divergence ranged from 0.12% to 1.22%, while interspecific distances ranged from 11.59% to 19.29%. Neighbor‐joining (NJ) analysis using the Kimura 2‐parameter model also showed specimens of the same species to be clustered together. The study provides the first cox1 sequences for three species of sand flies and indicates the utility of DNA barcoding for phlebotomine sand flies species identification in southeastern Mexico.     

Inter- and intra-specific characterization of tapeworms of the genus Diphyllobothrium (Cestoda: Diphyllobothriidea) from Switzerland,using nuclear and mitochondrial DNA targets

Human diphyllobothriosis is caused by at least 14 species of cestodes belonging to the genus Diphyllobothrium. Molecular analysis by sequencing of nuclear and mitochondrial targets identifies some species at inter- and intra-specific level, and helps to reconstruct their phylogenetic relationships. Nevertheless, the suitability of further molecular targets deserves to be widened, and the comparison of samples of different geographical origin could allow their intra-specific characterization, which could also be useful for epidemiological purposes. In this study, we investigated inter- and intra-specific variability among tapeworms of the genus Diphyllobothrium, with focus on Diphyllobothrium latum, originated from Switzerland. Samples were analyzed by comparing the sequences of two nuclear and two mitochondrial DNA targets. We analyzed 27 samples belonging to 4 species (D. latum, Diphyllobothrium nihonkaiense, Diphyllobothrium dendriticum and Diphyllobothrium ditremum), 15 of which isolated from clinical cases (adults and eggs), 2 from wild canines, and 2 from fish of Swiss lakes (plerocercoid larvae); 8 samples of homologous species from other geographic origins were also sequenced and compared with the Swiss ones. Sequences of partial small subunit ribosomal RNA (18S rRNA) gene and partial internal transcribed spacers 1 and 2 (ITS1-2) were not useful even in inter-specific identification, whereas sequences of complete cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cob) genes allowed us to assess inter- and intra-specific variations among the samples. Cox1 and cob could differentiate 3 and 5 haplotypes within the species D. latum. The results are discussed in the light of the anamneses provided by part of the patients.     

DNA splicing of mitochondrial group I and II introns in Schizosaccharomyces pombe

Summary In this paper we report the precise excision of the group I intron aI2b from the cox1 gene and of the group II intron bI from the cob gene fo the Schizosaccharomyces pombe strain 50. We present evidence that DNA excision of both intron DNA sequences is under nuclear control. Attempts to remove the first cox1 intron (aI1) have failed so far, but a deletion of approximately 200 bp in the open intronic reading frame demonstrates that it is not essential for normal cellular functions.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome c oxidase - cob gene encoding apocytochrome b - rns and rnl genes encoding the small and large ribosomal RNA - atp6, atp8 and atp9 genes encoding subunits 6, 8, and 9 of the ATP synthase complex - urfa unassigned reading frame a - aI1, aI2a, aI2b, aI3 introns in the cox 1 gene of S. pombe - bI intron in the cob gene - del-aI2b and del-bI respiratory competent strains in which the respective introns have been deleted by DNA splicing     

Identification of scuticociliates (Pseudocohnilembus persalinus,P. longisetus,Uronema marinum and Miamiensis avidus) based on the cox1 sequence

Scuticociliatosis is characterized as highly histophagous, causing systemic tissue destruction and high mortality in cultured marine fish. Some of the scuticociliates have been implicated as the causative agents of scuticociliatosis. Here, we describe our study to differentially identify various species in complex animal-sourced samples, namely olive flounder Paralichthys olivaceus and black rockfish Sebastes schlegelii suffering from scuticociliatosis. The mitochondrial cytochrome c oxidase 1 (cox1) gene from the scuticociliates was amplified and sequenced. The divergence percentage of small subunit ribosomal DNA sequence between average scuticociliate species was found to be low (8.3%) but the genetic divergence of cox1 sequence reached 23.5%, suggesting that a hyper-variable region of the cox1 gene could be used as a diagnostic DNA barcoding region. Thus, we developed species-specific primers for use in multiplex PCR of complex (pooled) samples. The primers yielded species-specific fragments (of distinct size) that allowed for simple, rapid, and effective identification and differentiation of multiple species present in a single sample.