Geographically widespread swordfish barcode stock identification: a case study of its application

Background

The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species.

Methodology

We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5′ hyper-variable fragment of the control region (5′dloop), were also used to validate the efficacy of COI as a stock-specific marker.

Case Report

This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products.

Conclusions

The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5′dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.     

Barcoding sponges: an overview based on comprehensive sampling

Background

Phylum Porifera includes ∼8,500 valid species distributed world-wide in aquatic ecosystems ranging from ephemeral fresh-water bodies to coastal environments and the deep-sea. The taxonomy and systematics of sponges is complicated, and morphological identification can be both time consuming and erroneous due to phenotypic convergence and secondary losses, etc. DNA barcoding can provide sponge biologists with a simple and rapid method for the identification of samples of unknown taxonomic membership. The Sponge Barcoding Project (www.spongebarcoding.org), the first initiative to barcode a non-bilaterian metazoan phylum, aims to provide a comprehensive DNA barcode database for Phylum Porifera.

Methodology/Principal Findings

∼7,400 sponge specimens have been extracted, and amplification of the standard COI barcoding fragment has been attempted for approximately 3,300 museum samples with ∼25% mean amplification success. Based on this comprehensive sampling, we present the first report on the workflow and progress of the sponge barcoding project, and discuss some common pitfalls inherent to the barcoding of sponges.

Conclusion

A DNA-barcoding workflow capable of processing potentially large sponge collections has been developed and is routinely used for the Sponge Barcoding Project with success. Sponge specific problems such as the frequent co-amplification of non-target organisms have been detected and potential solutions are currently under development. The initial success of this innovative project have already demonstrated considerable refinement of sponge systematics, evaluating morphometric character importance, geographic phenotypic variability, and the utility of the standard barcoding fragment for Porifera (despite its conserved evolution within this basal metazoan phylum).     

Intraspecific Inversions Pose a Challenge for the trnH-psbA Plant DNA Barcode

Background

The chloroplast trnH-psbA spacer region has been proposed as a prime candidate for use in DNA barcoding of plants because of its high substitution rate. However, frequent inversions associated with palindromic sequences within this region have been found in multiple lineages of Angiosperms and may complicate its use as a barcode, especially if they occur within species.

Methodology/Principal Findings

Here, we evaluate the implications of intraspecific inversions in the trnH-psbA region for DNA barcoding efforts. We report polymorphic inversions within six species of Gentianaceae, all narrowly circumscribed morphologically: Gentiana algida, Gentiana fremontii, Gentianopsis crinita, Gentianopsis thermalis, Gentianopsis macrantha and Frasera speciosa. We analyze these sequences together with those from 15 other species of Gentianaceae and show that typical simple methods of sequence alignment can lead to misassignment of conspecifics and incorrect assessment of relationships.

Conclusions/Significance

Frequent inversions in the trnH-psbA region, if not recognized and aligned appropriately, may lead to large overestimates of the number of substitution events separating closely related lineages and to uniting more distantly related taxa that share the same form of the inversion. Thus, alignment of the trnH-psbA spacer region will need careful attention if it is used as a marker for DNA barcoding.     

Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/Principal Findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/Significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.     

Oligonucleotide Frequencies of Barcoding Loci Can Discriminate Species across Kingdoms

Background

DNA barcoding refers to the use of short DNA sequences for rapid identification of species. Genetic distance or character attributes of a particular barcode locus discriminate the species. We report an efficient approach to analyze short sequence data for discrimination between species.

Methodology and Principal Findings

A new approach, Oligonucleotide Frequency Range (OFR) of barcode loci for species discrimination is proposed. OFR of the loci that discriminates between species was characteristic of a species, i.e., the maxima and minima within a species did not overlap with that of other species. We compared the species resolution ability of different barcode loci using p-distance, Euclidean distance of oligonucleotide frequencies, nucleotide-character based approach and OFR method. The species resolution by OFR was either higher or comparable to the other methods. A short fragment of 126 bp of internal transcribed spacer region in ribosomal RNA gene was sufficient to discriminate a majority of the species using OFR.

Conclusions/Significance

Oligonucleotide frequency range of a barcode locus can discriminate between species. Ability to discriminate species using very short DNA fragments may have wider applications in forensic and conservation studies.     

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.     

A comprehensive DNA barcode library for the looper moths (Lepidoptera: Geometridae) of British Columbia, Canada

Background

The construction of comprehensive reference libraries is essential to foster the development of DNA barcoding as a tool for monitoring biodiversity and detecting invasive species. The looper moths of British Columbia (BC), Canada present a challenging case for species discrimination via DNA barcoding due to their considerable diversity and limited taxonomic maturity.

Methodology/Principal Findings

By analyzing specimens held in national and regional natural history collections, we assemble barcode records from representatives of 400 species from BC and surrounding provinces, territories and states. Sequence variation in the barcode region unambiguously discriminates over 93% of these 400 geometrid species. However, a final estimate of resolution success awaits detailed taxonomic analysis of 48 species where patterns of barcode variation suggest cases of cryptic species, unrecognized synonymy as well as young species.

Conclusions/Significance

A catalog of these taxa meriting further taxonomic investigation is presented as well as the supplemental information needed to facilitate these investigations.     

Beyond the colours: discovering hidden diversity in the Nymphalidae of the Yucatan Peninsula in Mexico through DNA barcoding

Background

Recent studies have demonstrated the utility of DNA barcoding in the discovery of overlooked species and in the connection of immature and adult stages. In this study, we use DNA barcoding to examine diversity patterns in 121 species of Nymphalidae from the Yucatan Peninsula in Mexico. Our results suggest the presence of cryptic species in 8 of these 121 taxa. As well, the reference database derived from the analysis of adult specimens allowed the identification of nymphalid caterpillars providing new details on host plant use.

Methodology/Principal Findings

We gathered DNA barcode sequences from 857 adult Nymphalidae representing 121 different species. This total includes four species (Adelpha iphiclus, Adelpha malea, Hamadryas iphtime and Taygetis laches) that were initially overlooked because of their close morphological similarity to other species. The barcode results showed that each of the 121 species possessed a diagnostic array of barcode sequences. In addition, there was evidence of cryptic taxa; seven species included two barcode clusters showing more than 2% sequence divergence while one species included three clusters. All 71 nymphalid caterpillars were identified to a species level by their sequence congruence to adult sequences. These caterpillars represented 16 species, and included Hamadryas julitta, an endemic species from the Yucatan Peninsula whose larval stages and host plant (Dalechampia schottii, also endemic to the Yucatan Peninsula) were previously unknown.

Conclusions/Significance

This investigation has revealed overlooked species in a well-studied museum collection of nymphalid butterflies and suggests that there is a substantial incidence of cryptic species that await full characterization. The utility of barcoding in the rapid identification of caterpillars also promises to accelerate the assembly of information on life histories, a particularly important advance for hyperdiverse tropical insect assemblages.     

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.     

Monophyly,Distance and Character–Based Multigene Barcoding Reveal Extraordinary Cryptic Diversity in Nassarius: A Complex and Dangerous Community

Background

Correct identification and cryptic biodiversity revelation for marine organisms are pressing since the marine life is important in maintaining the balance of ecological system and is facing the problem of biodiversity crisis or food safety. DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Nassarius, the common mudsnail, plays an important role in marine environment and has problem in food safety, but the classification of it is quite confused because of the complex morphological diversity.

Methodology/Principal Findings

Here we report a comprehensive barcoding analysis of 22 Nassarius species. We integrated the mitochondrial and nuclear sequences and the morphological characters to determine 13 Nassarius species studied and reveal four cryptic species and one pair synonyms. Distance, monophyly, and character–based barcoding methods were employed.

Conclusions/Significance

Such successful identification and unexpected cryptic discovery is significant for Nassarius in food safety and species conversation and remind us to pay more attention to the hidden cryptic biodiversity ignored in marine life. Distance, monophyly, and character–based barcoding methods are all very helpful in identification but the character-based method shows some advantages.     

DNA Barcoding of Shark Meats Identify Species Composition and CITES-Listed Species from the Markets in Taiwan

Background

An increasing awareness of the vulnerability of sharks to exploitation by shark finning has contributed to a growing concern about an unsustainable shark fishery. Taiwan’s fleet has the 4th largest shark catch in the world, accounting for almost 6% of the global figures. Revealing the diversity of sharks consumed by Taiwanese is important in designing conservation plans. However, fins make up less than 5% of the total body weight of a shark, and their bodies are sold as filets in the market, making it difficult or impossible to identify species using morphological traits.

Methods

In the present study, we adopted a DNA barcoding technique using a 391-bp fragment of the mitochondrial cytochrome oxidase I (COI) gene to examine the diversity of shark filets and fins collected from markets and restaurants island-wide in Taiwan.

Results

Amongst the 548 tissue samples collected and sequenced, 20 major clusters were apparent by phylogenetic analyses, each of them containing individuals belonging to the same species (most with more than 95% bootstrap values), corresponding to 20 species of sharks. Additionally, Alopias pelagicus, Carcharhinus falciformis, Isurus oxyrinchus, and Prionace glauca consisted of 80% of the samples we collected, indicating that these species might be heavily consumed in Taiwan. Approximately 5% of the tissue samples used in this study were identified as species listed in CITES Appendix II, including two species of Sphyrna, C. longimanus and Carcharodon carcharias.

Conclusion

DNA barcoding provides an alternative method for understanding shark species composition when species-specific data is unavailable. Considering the global population decline, stock assessments of Appendix II species and highly consumed species are needed to accomplish the ultimate goal of shark conservation.     

A Validated Methodology for Genetic Identification of Tuna Species (Genus Thunnus)

Background

Tuna species of the genus Thunnus, such as the bluefin tunas, are some of the most important and yet most endangered trade fish in the world. Identification of these species in traded forms, however, may be difficult depending on the presentation of the products, which may hamper conservation efforts on trade control. In this paper, we validated a genetic methodology that can fully distinguish between the eight Thunnus species from any kind of processed tissue.

Methodology

After testing several genetic markers, a complete discrimination of the eight tuna species was achieved using Forensically Informative Nucleotide Sequencing based primarily on the sequence variability of the hypervariable genetic marker mitochondrial DNA control region (mtDNA CR), followed, in some specific cases, by a second validation by a nuclear marker rDNA first internal transcribed spacer (ITS1). This methodology was able to distinguish all tuna species, including those belonging to the subgenus Neothunnus that are very closely related, and in consequence can not be differentiated with other genetic markers of lower variability. This methodology also took into consideration the presence of introgression that has been reported in past studies between T. thynnus, T. orientalis and T. alalunga. Finally, we applied the methodology to cross-check the species identity of 26 processed tuna samples.

Conclusions

Using the combination of two genetic markers, one mitochondrial and another nuclear, allows a full discrimination between all eight tuna species. Unexpectedly, the genetic marker traditionally used for DNA barcoding, cytochrome oxidase 1, could not differentiate all species, thus its use as a genetic marker for tuna species identification is questioned.     

DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

Background

Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported.

Methodology/Principal Findings

We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases.

Conclusions/Significance

This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.     

DNA barcoding identifies Argentine fishes from marine and brackish waters

Background

DNA barcoding has been advanced as a promising tool to aid species identification and discovery through the use of short, standardized gene targets. Despite extensive taxonomic studies, for a variety of reasons the identification of fishes can be problematic, even for experts. DNA barcoding is proving to be a useful tool in this context. However, its broad application is impeded by the need to construct a comprehensive reference sequence library for all fish species. Here, we make a regional contribution to this grand challenge by calibrating the species discrimination efficiency of barcoding among 125 Argentine fish species, representing nearly one third of the known fauna, and examine the utility of these data to address several key taxonomic uncertainties pertaining to species in this region.

Methodology/Principal Findings

Specimens were collected and morphologically identified during crusies conducted between 2005 and 2008. The standard BARCODE fragment of COI was amplified and bi-directionally sequenced from 577 specimens (mean of 5 specimens/species), and all specimens and sequence data were archived and interrogated using analytical tools available on the Barcode of Life Data System (BOLD; www.barcodinglife.org). Nearly all species exhibited discrete clusters of closely related haplogroups which permitted the discrimination of 95% of the species (i.e. 119/125) examined while cases of shared haplotypes were detected among just three species-pairs. Notably, barcoding aided the identification of a new species of skate, Dipturus argentinensis, permitted the recognition of Genypterus brasiliensis as a valid species and questions the generic assignment of Paralichthys isosceles.

Conclusions/Significance

This study constitutes a significant contribution to the global barcode reference sequence library for fishes and demonstrates the utility of barcoding for regional species identification. As an independent assessment of alpha taxonomy, barcodes provide robust support for most morphologically based taxon concepts and also highlight key areas of taxonomic uncertainty worthy of reappraisal.     

trnL-F is a powerful marker for DNA identification of field vittarioid gametophytes (Pteridaceae)

Background and Aims

The gametophyte phase of ferns plays an important role in habitat selection, dispersal, adaptation and evolution. However, ecological studies on fern gametophytes have been impeded due to the difficulty of species identification of free-living gametophytes. DNA barcoding provides an alternative approach to identifying fern gametophytes but is rarely applied to field studies. In this study, an example of field vittarioid gametophyte identification using DNA barcoding, which has not been done before, is given.

Methods

A combination of distance-based and tree-based approaches was performed to evaluate the discriminating power of three candidate barcodes (matK, rbcL and trnL-F) on 16 vittarioid sporophytes. Sequences of the trnL-F region were generated from 15 fern gametophyte populations by tissue-direct PCR and were compared against the sporophyte dataset, using BLAST.

Key Results trnL-F

earns highest primer universality and discriminatory ability scores, whereas PCR success rates were very low for matK and rbcL regions (10·8 % and 41·3 %, respectively). BLAST analyses showed that all the sampled field gametophytes could be successfully identified to species level. Three gametophyte populations were also discovered to be living beyond the known occurrence of their sporophyte counterparts.

Conclusions

This study demonstrates that DNA barcoding (i.e. reference databasing, tissue-direct PCR and molecular analysis), especially the trnL-F region, is an efficient tool to identify field gametophytes, and has considerable potential in exploring the ecology of fern gametophytes.     

Molecular approach to the identification of fish in the South China Sea

Background

DNA barcoding is one means of establishing a rapid, accurate, and cost-effective system for the identification of species. It involves the use of short, standard gene targets to create sequence profiles of known species against sequences of unknowns that can be matched and subsequently identified. The Fish Barcode of Life (FISH-BOL) campaign has the primary goal of gathering DNA barcode records for all the world''s fish species. As a contribution to FISH-BOL, we examined the degree to which DNA barcoding can discriminate marine fishes from the South China Sea.

Methodology/Principal Findings

DNA barcodes of cytochrome oxidase subunit I (COI) were characterized using 1336 specimens that belong to 242 species fishes from the South China Sea. All specimen provenance data (including digital specimen images and geospatial coordinates of collection localities) and collateral sequence information were assembled using Barcode of Life Data System (BOLD; www.barcodinglife.org). Small intraspecific and large interspecific differences create distinct genetic boundaries among most species. In addition, the efficiency of two mitochondrial genes, 16S rRNA (16S) and cytochrome b (cytb), and one nuclear ribosomal gene, 18S rRNA (18S), was also evaluated for a few select groups of species.

Conclusions/Significance

The present study provides evidence for the effectiveness of DNA barcoding as a tool for monitoring marine biodiversity. Open access data of fishes from the South China Sea can benefit relative applications in ecology and taxonomy.     

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

Background

The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over.

Methodology/Principal Findings

Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level.

Conclusions

The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.     

Ecological niche modelling and nDNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding

Background

DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data.

Methodology/Principal Findings

The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n.

Conclusion/Significance

In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species.     

How Effective Are DNA Barcodes in the Identification of African Rainforest Trees?

Background

DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species.

Methodology/Principal Findings

We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95–100% success), but less for species identification (71–88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84–90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA.

Conclusions/Significance

Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications.