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.     

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.     

CO I Barcoding Reveals New Clades and Radiation Patterns of Indo-Pacific Sponges of the Family Irciniidae (Demospongiae: Dictyoceratida)

Background

DNA barcoding is a promising tool to facilitate a rapid and unambiguous identification of sponge species. Demosponges of the order Dictyoceratida are particularly challenging to identify, but are of ecological as well as biochemical importance.

Methodology/Principal Findings

Here we apply DNA barcoding with the standard CO1-barcoding marker on selected Indo-Pacific specimens of two genera, Ircinia and Psammocinia of the family Irciniidae. We show that the CO1 marker identifies several species new to science, reveals separate radiation patterns of deep-sea Ircinia sponges and indicates dispersal patterns of Psammocinia species. However, some species cannot be unambiguously barcoded by solely this marker due to low evolutionary rates.

Conclusions/Significance

We support previous suggestions for a combination of the standard CO1 fragment with an additional fragment for sponge DNA barcoding.     

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.     

The Diversity of Coolia spp. (Dinophyceae Ostreopsidaceae) in the Central Great Barrier Reef Region

Background

Dinoflagellates are important primary producers, crucial in marine food webs. Toxic strains, however, are the main causative agents of non-bacterial seafood poisoning, a major concern for public health worldwide. Despite their importance, taxonomic uncertainty within many genera of dinoflagellates is still high. The genus Coolia includes potentially harmful species and the diversity within the genus is just starting to become apparent.

Methodology/Principal Findings

In the current study, cultures were established from strains of Coolia spp. isolated from the central Great Barrier Reef (GBR). Cultures were identified based on thecal plate morphology and analyses of sequences (18S, ITS and 28S) from the nuclear rRNA operon. We report that the central GBR harbors a high diversity of Coolia species, including two species known to be capable of toxin production (C. tropicalis and C. malayensis), as well as the non-toxic C. canariensis. The strain of C. canariensis isolated from the GBR may in fact be a cryptic species, closely related but nevertheless phylogenetically distinct from the strain on which the holotype of C. canariensis was based. We also found evidence of the occurrence of a cryptic species morphologically very similar to both C. malayensis and C. monotis. The consequences of taxonomic confusion within the genus are discussed.

Conclusion/Significance

The central GBR region harbors a previously unreported high diversity of Coolia spp., including two species known to potentially produce toxins. The presence of a cryptic species of unknown toxicity highlights the importance of cryptic diversity within dinoflagellates.     

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.     

Environmental barcoding reveals massive dinoflagellate diversity in marine environments

Background

Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments.

Methodology/Principal Findings

In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species.

Conclusions/Significance

COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.     

Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda)

Background

Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied.

Methodology/Principal Findings

We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies.

Conclusions/Significance

Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.     

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA

Background

Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.

Methodology/Principal Findings

In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.

Conclusions

Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.     

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.     

Identification of Clinically Relevant Fungi and Prototheca Species by rRNA Gene Sequencing and Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

Background

Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.

Methods

One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5′ end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.

Results

For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.

Conclusions

rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.     

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 Reveals Limited Accuracy of Identifications Based on Folk Taxonomy

Background

The trade of plant roots as traditional medicine is an important source of income for many people around the world. Destructive harvesting practices threaten the existence of some plant species. Harvesters of medicinal roots identify the collected species according to their own folk taxonomies, but once the dried or powdered roots enter the chain of commercialization, accurate identification becomes more challenging.

Methodology

A survey of morphological diversity among four root products traded in the medina of Marrakech was conducted. Fifty-one root samples were selected for molecular identification using DNA barcoding using three markers, trnH-psbA, rpoC1, and ITS. Sequences were searched using BLAST against a tailored reference database of Moroccan medicinal plants and their closest relatives submitted to NCBI GenBank.

Principal Findings

Combining psbA-trnH, rpoC1, and ITS allowed the majority of the market samples to be identified to species level. Few of the species level barcoding identifications matched the scientific names given in the literature, including the most authoritative and widely cited pharmacopeia.

Conclusions/Significance

The four root complexes selected from the medicinal plant products traded in Marrakech all comprise more than one species, but not those previously asserted. The findings have major implications for the monitoring of trade in endangered plant species as morphology-based species identifications alone may not be accurate. As a result, trade in certain species may be overestimated, whereas the commercialization of other species may not be recorded at all.     

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.     

Australian Sphingidae – DNA Barcodes Challenge Current Species Boundaries and Distributions

Main Objective

We examine the extent of taxonomic and biogeographical uncertainty in a well-studied group of Australian Lepidoptera, the hawkmoths (Sphingidae).

Methods

We analysed the diversity of Australian sphingids through the comparative analysis of their DNA barcodes, supplemented by morphological re-examinations and sequence information from a nuclear marker in selected cases. The results from the analysis of Australian sphingids were placed in a broader context by including conspecifics and closely related taxa from outside Australia to test taxonomic boundaries.

Results

Our results led to the discovery of six new species in Australia, one case of erroneously synonymized species, and three cases of synonymy. As a result, we establish the occurrence of 75 species of hawkmoths on the continent. The analysis of records from outside Australia also challenges the validity of current taxonomic boundaries in as many as 18 species, including Agrius convolvuli (Linnaeus, 1758), a common species that has gained adoption as a model system. Our work has revealed a higher level of endemism than previously recognized. Most (90%) Australian sphingids are endemic to the continent (45%) or to Australia, the Pacific Islands and the Papuan and Wallacean regions (45%). Only seven species (10%) have ranges that extend beyond this major biogeographical boundary toward SE Asia and other regions of the Old World.

Main Conclusions

This study has established that overlooked cryptic diversity and inaccurate species delineation produced significant misconceptions concerning diversity and distribution patterns in a group of insects that is considered well known taxonomically. Because DNA barcoding represents a straightforward way to test taxonomic boundaries, its implementation can improve the accuracy of primary diversity data in biogeography and conservation studies.     

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.     

The Internal Transcribed Spacer (ITS) Region and trnhH-psbA Are Suitable Candidate Loci for DNA Barcoding of Tropical Tree Species of India

Background

DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. rbcL and matK are standard barcode loci while ITS, and trnH-psbA are considered as supplementary loci for plants.

Methodology and Principal Findings

Plant barcode loci, namely, rbcL, matK, ITS, trnH-psbA, and the recently proposed ITS2, were tested for their efficacy as barcode loci using 300 accessions of tropical tree species. We tested these loci for PCR, sequencing success, and species discrimination ability using three methods. rbcL was the best locus as far as PCR and sequencing success rate were concerned, but not for the species discrimination ability of tropical tree species. ITS and trnH-psbA were the second best loci in PCR and sequencing success, respectively. The species discrimination ability of ITS ranged from 24.4 percent to 74.3 percent and that of trnH-psbA was 25.6 percent to 67.7 percent, depending upon the data set and the method used. matK provided the least PCR success, followed by ITS2 (59. 0%). Species resolution by ITS2 and rbcL ranged from 9.0 percent to 48.7 percent and 13.2 percent to 43.6 percent, respectively. Further, we observed that the NCBI nucleotide database is poorly represented by the sequences of barcode loci studied here for tree species.

Conclusion

Although a conservative approach of a success rate of 60–70 percent by both ITS and trnH-psbA may not be considered as highly successful but would certainly help in large-scale biodiversity inventorization, particularly for tropical tree species, considering the standard success rate of plant DNA barcode program reported so far. The recommended matK and rbcL primers combination may not work in tropical tree species as barcode markers.     

The Inadequacy of Morphology for Species and Genus Delineation in Microbial Eukaryotes: An Example from the Parabasalian Termite Symbiont Coronympha

Background

For the majority of microbial eukaryotes (protists, algae), there is no clearly superior species concept that is consistently applied. In the absence of a practical biological species concept, most species and genus level delineations have historically been based on morphology, which may lead to an underestimate of the diversity of microbial eukaryotes. Indeed, a growing body of molecular evidence, such as barcoding surveys, is beginning to support the conclusion that significant cryptic species diversity exists. This underestimate of diversity appears to be due to a combination of using morphology as the sole basis for assessing diversity and our inability to culture the vast majority of microbial life. Here we have used molecular markers to assess the species delineations in two related but morphologically distinct genera of uncultivated symbionts found in the hindgut of termites.

Methodology/Principal Findings

Using single-cell isolation and environmental PCR, we have used a barcoding approach to characterize the diversity of Coronympha and Metacoronympha symbionts in four species of Incisitermes termites, which were also examined using scanning electron microscopy and light microcopy. Despite the fact that these genera are significantly different in morphological complexity and structural organisation, we find they are two life history stages of the same species. At the same time, we show that the symbionts from different termite hosts show an equal or greater level of sequence diversity than do the hosts, despite the fact that the symbionts are all classified as one species.

Conclusions/Significance

The morphological information used to describe the diversity of these microbial symbionts is misleading at both the genus and species levels, and led to an underestimate of species level diversity as well as an overestimate of genus level diversity. The genus ‘Metacoronympha’ is invalid and appears to be a life history stage of Coronympha, while the single recognized species of Coronympha octonaria inhabiting these four termites is better described as four distinct species.     

Barcoding Poplars (Populus L.) from Western China

Background

Populus is an ecologically and economically important genus of trees, but distinguishing between wild species is relatively difficult due to extensive interspecific hybridization and introgression, and the high level of intraspecific morphological variation. The DNA barcoding approach is a potential solution to this problem.

Methodology/Principal Findings

Here, we tested the discrimination power of five chloroplast barcodes and one nuclear barcode (ITS) among 95 trees that represent 21 Populus species from western China. Among all single barcode candidates, the discrimination power is highest for the nuclear ITS, progressively lower for chloroplast barcodes matK (M), trnG-psbK (G) and psbK-psbI (P), and trnH-psbA (H) and rbcL (R); the discrimination efficiency of the nuclear ITS (I) is also higher than any two-, three-, or even the five-locus combination of chloroplast barcodes. Among the five combinations of a single chloroplast barcode plus the nuclear ITS, H+I and P+I differentiated the highest and lowest portion of species, respectively. The highest discrimination rate for the barcodes or barcode combinations examined here is 55.0% (H+I), and usually discrimination failures occurred among species from sympatric or parapatric areas.

Conclusions/Significance

In this case study, we showed that when discriminating Populus species from western China, the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions. Meanwhile, combining the ITS region with chloroplast regions may improve the barcoding success rate and assist in detecting recent interspecific hybridizations. Failure to discriminate among several groups of Populus species from sympatric or parapatric areas may have been the result of incomplete lineage sorting, frequent interspecific hybridizations and introgressions. We agree with a previous proposal for constructing a tiered barcoding system in plants, especially for taxonomic groups that have complex evolutionary histories (e.g. Populus).     

Application of the ITS2 Region for Barcoding Medicinal Plants of Selaginellaceae in Pteridophyta

Background

Selaginellaceae is a family of nonseed plants with special evolutionary significance. Plants of the family Selaginellaceae are similarly shaped and easily confused, complicating identification via traditional methods. This study explored, for the first time, the use of the DNA barcode ITS2 to identify medicinal plants of the Selaginellaceae family.

Methodology/Principal Findings

In our study, 103 samples were collected from the main distribution areas in China; these samples represented 34 species and contained almost all of the medicinal plants of Selaginellaceae. The ITS2 region of the genome was amplified from these samples and sequenced using universal primers and reaction conditions. The success rates of the PCR amplification and sequencing were 100%. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 regions, while the presence of a barcoding gap was obvious. Using the BLAST1 and nearest distance methods, our results proved that the ITS2 regions could successfully identify the species of all Selaginellaceae samples examined. In addition, the secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Selaginellaceae. Furthermore, cluster analysis using the ITS2 barcode supported the relationship between the species of Selaginellaceae established by traditional morphological methods.

Conclusion

The ITS2 barcode can effectively identify medicinal plants of Selaginellaceae. The results provide a scientific basis for the precise identification of plants of the family Selaginellaceae and the reasonable development of these resources. This study may broaden the application of DNA barcoding in the medicinal plant field and benefit phylogenetic investigations.