Testing a short nuclear marker for inferring staphylinid beetle diversity in an African tropical rain forest

Background

The use of DNA based methods for assessing biodiversity has become increasingly common during the last years. Especially in speciose biomes as tropical rain forests and/or in hyperdiverse or understudied taxa they may efficiently complement morphological approaches. The most successful molecular approach in this field is DNA barcoding based on cytochrome c oxidase I (COI) marker, but other markers are used as well. Whereas most studies aim at identifying or describing species, there are only few attempts to use DNA markers for inventorying all animal species found in environmental samples to describe variations of biodiversity patterns.

Methodology/Principal Findings

In this study, an analysis of the nuclear D3 region of the 28S rRNA gene to delimit species-like units is compared to results based on distinction of morphospecies. Data derived from both approaches are used to assess diversity and composition of staphylinid beetle communities of a Guineo-Congolian rain forest in Kenya. Beetles were collected with a standardized sampling design across six transects in primary and secondary forests using pitfall traps. Sequences could be obtained of 99% of all individuals. In total, 76 molecular operational taxonomic units (MOTUs) were found in contrast to 70 discernible morphospecies. Despite this difference both approaches revealed highly similar biodiversity patterns, with species richness being equal in primary and secondary forests, but with divergent species communities in different habitats. The D3-MOTU approach proved to be an efficient tool for biodiversity analyses.

Conclusions/Significance

Our data illustrate that the use of MOTUs as a proxy for species can provide an alternative to morphospecies identification for the analysis of changes in community structure of hyperdiverse insect taxa. The efficient amplification of the D3-marker and the ability of the D3-MOTUs to reveal similar biodiversity patterns as analyses of morphospecies recommend its use in future molecular studies on biodiversity.     

DNA barcoding for community ecology--how to tackle a hyperdiverse, mostly undescribed Melanesian fauna

Background

Trigonopterus weevils are widely distributed throughout Melanesia and hyperdiverse in New Guinea. They are a dominant feature in natural forests, with narrow altitudinal zonation. Their use in community ecology has been precluded by the “taxonomic impediment”.

Methodology/Principal Findings

We sampled >6,500 specimens from seven areas across New Guinea; 1,002 specimens assigned to 270 morphospecies were DNA sequenced. Objective clustering of a refined dataset (excluding nine cryptic species) at 3% threshold revealed 324 genetic clusters (DNA group count relative to number of morphospecies = 20.0% overestimation of species diversity, or 120.0% agreement) and 85.6% taxonomic accuracy (the proportion of DNA groups that “perfectly” agree with morphology-based species hypotheses). Agreement and accuracy were best at an 8% threshold. GMYC analysis revealed 328 entities (21.5% overestimation) with 227 perfect GMYC entities (84.1% taxonomic accuracy). Both methods outperform the parataxonomist (19% underestimation; 31.6% taxonomic accuracy). The number of species found in more than one sampling area was highest in the Eastern Highlands and Huon (Sørensen similarity index 0.07, 4 shared species); ⅓ of all areas had no species overlap. Success rates of DNA barcoding methods were lowest when species showed a pronounced geographical structure. In general, Trigonopterus show high α and β-diversity across New Guinea.

Conclusions/Significance

DNA barcoding is an excellent tool for biodiversity surveys but success rates might drop when closer localities are included. Hyperdiverse Trigonopterus are a useful taxon for evaluating forest remnants in Melanesia, allowing finer-grained analyses than would be possible with vertebrate taxa commonly used to date. Our protocol should help establish other groups of hyperdiverse fauna as target taxa for community ecology. Sequencing delivers objective data on taxa of incredible diversity but mostly without a solid taxonomic foundation and should help pave the road for the eventual formal naming of new species.     

A Good Compromise: Rapid and Robust Species Proxies for Inventorying Biodiversity Hotspots Using the Terebridae (Gastropoda: Conoidea)

Devising a reproducible approach for species delimitation of hyperdiverse groups is an ongoing challenge in evolutionary biology. Speciation processes combine modes of passive and adaptive trait divergence requiring an integrative taxonomy approach to accurately generate robust species hypotheses. However, in light of the rapid decline of diversity on Earth, complete integrative approaches may not be practical in certain species-rich environments. As an alternative, we applied a two-step strategy combining ABGD (Automated Barcode Gap Discovery) and Klee diagrams, to balance speed and accuracy in producing primary species hypotheses (PSHs). Specifically, an ABGD/Klee approach was used for species delimitation in the Terebridae, a neurotoxin-producing marine snail family included in the Conoidea. Delimitation of species boundaries is problematic in the Conoidea, as traditional taxonomic approaches are hampered by the high levels of variation, convergence and morphological plasticity of shell characters. We used ABGD to analyze gaps in the distribution of pairwise distances of 454 COI sequences attributed to 87 morphospecies and obtained 98 to 125 Primary Species Hypotheses (PSHs). The PSH partitions were subsequently visualized as a Klee diagram color map, allowing easy detection of the incongruences that were further evaluated individually with two other species delimitation models, General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). GMYC and PTP results confirmed the presence of 17 putative cryptic terebrid species in our dataset. The consensus of GMYC, PTP, and ABGD/Klee findings suggest the combination of ABGD and Klee diagrams is an effective approach for rapidly proposing primary species proxies in hyperdiverse groups and a reliable first step for macroscopic biodiversity assessment.     

How DNA barcodes complement taxonomy and explore species diversity: the case study of a poorly understood marine fauna

Background

The species boundaries of some venerids are difficult to define based solely on morphological features due to their indistinct intra- and interspecific phenotypic variability. An unprecedented biodiversity crisis caused by human activities has emerged. Thus, to access the biological diversity and further the conservation of this taxonomically muddling bivalve group, a fast and simple approach that can efficiently examine species boundaries and highlight areas of unrecognized diversity is urgently needed. DNA barcoding has proved its effectiveness in high-volume species identification and discovery. In the present study, Chinese fauna was chosen to examine whether this molecular biomarker is sensitive enough for species delimitation, and how it complements taxonomy and explores species diversity.

Methodology/Principal Findings

A total of 315 specimens from around 60 venerid species were included, qualifying the present study as the first major analysis of DNA barcoding for marine bivalves. Nearly all individuals identified to species level based on morphological traits possessed distinct barcode clusters, except for the specimens of one species pair. Among the 26 individuals that were not assigned binomial names a priori, twelve respectively nested within a species genealogy. The remaining individuals formed five monophyletic clusters that potentially represent species new to science or at least unreported in China. Five putative hidden species were also uncovered in traditional morphospecies.

Conclusions/Significance

The present study shows that DNA barcoding is effective in species delimitation and can aid taxonomists by indicating useful diagnostic morphological traits, informing needful revision, and flagging unseen species. Moreover, the BOLD system, which deposits barcodes, morphological, geographical and other data, has the potential as a convenient taxonomic platform.     

Excluding spatial sampling bias does not eliminate oversplitting in DNA‐based species delimitation analyses

DNA barcoding and DNA‐based species delimitation are major tools in DNA taxonomy. Sampling has been a central debate in this context, because the geographical composition of samples affects the accuracy and performance of DNA barcoding. Performance of complex DNA‐based species delimitation is to be tested under simpler conditions in absence of geographic sampling bias. Here, we present an empirical dataset sampled from a single locality in a Southeast‐Asian biodiversity hotspot (Laos: Phou Pan mountain). We investigate the performance of various species delimitation approaches on a megadiverse assemblage of herbivorous chafer beetles (Coleoptera: Scarabaeidae) to infer whether species delimitation suffers in the same way from exaggerate infraspecific variation despite the lack of geographic genetic variation that led to inconsistencies between entities from DNA‐based and morphology‐based species inference in previous studies. For this purpose, a 658 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) was analyzed for a total of 186 individuals of 56 morphospecies. Tree‐based and distance‐based species delimitation methods were used. All approaches showed a rather limited match ratio (max. 77%) with morphospecies. Poisson tree process (PTP) and statistical parsimony network analysis (TCS) prevailingly over‐splitted morphospecies, while 3% clustering and Automatic Barcode Gap Discovery (ABGD) also lumped several species into one entity. ABGD revealed the highest congruence between molecular operational taxonomic units (MOTUs) and morphospecies. Disagreements between morphospecies and MOTUs have to be explained by historically acquired geographic genetic differentiation, incomplete lineage sorting, and hybridization. The study once again highlights how important morphology still is in order to correctly interpret the results of molecular species delimitation.     

Elevational gradients in bird diversity in the Eastern Himalaya: an evaluation of distribution patterns and their underlying mechanisms

Background

Understanding diversity patterns and the mechanisms underlying those patterns along elevational gradients is critically important for conservation efforts in montane ecosystems, especially those that are biodiversity hotspots. Despite recent advances, consensus on the underlying causes, or even the relative influence of a suite of factors on elevational diversity patterns has remained elusive.

Methods and Principal Findings

We examined patterns of species richness, density and range size distribution of birds, and the suite of biotic and abiotic factors (primary productivity, habitat variables, climatic factors and geometric constraints) that governs diversity along a 4500-m elevational gradient in the Eastern Himalayan region, a biodiversity hotspot within the world''s tallest mountains. We used point count methods for sampling birds and quadrats for estimating vegetation at 22 sites along the elevational gradient. We found that species richness increased to approximately 2000 m, then declined. We found no evidence that geometric constraints influenced this pattern, whereas actual evapotranspiration (a surrogate for primary productivity) and various habitat variables (plant species richness, shrub density and basal area of trees) accounted for most of the variation in bird species richness. We also observed that ranges of most bird species were narrow along the elevation gradient. We find little evidence to support Rapoport''s rule for the birds of Sikkim region of the Himalaya.

Conclusions and Significance

This study in the Eastern Himalaya indicates that species richness of birds is highest at intermediate elevations along one of the most extensive elevational gradients ever examined. Additionally, primary productivity and factors associated with habitat accounted for most of the variation in avian species richness. The diversity peak at intermediate elevations and the narrow elevational ranges of most species suggest important conservation implications: not only should mid-elevation areas be conserved, but the entire gradient requires equal conservation attention.     

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.     

Identification of Amazonian Trees with DNA Barcodes

Background

Large-scale plant diversity inventories are critical to develop informed conservation strategies. However, the workload required for classic taxonomic surveys remains high and is particularly problematic for megadiverse tropical forests.

Methodology/Principal Findings

Based on a comprehensive census of all trees in two hectares of a tropical forest in French Guiana, we examined whether plant DNA barcoding could contribute to increasing the quality and the pace of tropical plant biodiversity surveys. Of the eight plant DNA markers we tested (rbcLa, rpoC1, rpoB, matK, ycf5, trnL, psbA-trnH, ITS), matK and ITS had a low rate of sequencing success. More critically, none of the plastid markers achieved a rate of correct plant identification greater than 70%, either alone or combined. The performance of all barcoding markers was noticeably low in few species-rich clades, such as the Laureae, and the Sapotaceae. A field test of the approach enabled us to detect 130 molecular operational taxonomic units in a sample of 252 juvenile trees. Including molecular markers increased the identification rate of juveniles from 72% (morphology alone) to 96% (morphology and molecular) of the individuals assigned to a known tree taxon.

Conclusion/Significance

We conclude that while DNA barcoding is an invaluable tool for detecting errors in identifications and for identifying plants at juvenile stages, its limited ability to identify collections will constrain the practical implementation of DNA-based tropical plant biodiversity programs.     

Turnover and nestedness in subtropical dung beetle assemblages along an elevational gradient

Aim

We investigated changes in dung beetle β‐diversity components along a subtropical elevational gradient, to test whether turnover or nestedness‐related processes drive the dissimilarity of assemblages at spatial and temporal scales.

Location

An elevational gradient (200–1,600 m a.s.l.) of the Atlantic Forest in southern Brazil.

Methods

We investigated the extent to which β‐diversity varied along the elevational gradient (six elevations) at both spatial (among sites at different elevations) and temporal (different months at the same site) scales. We compared both the turnover and nestedness‐related dissimilarity of species and genera using multiple‐site or multiple‐month measures and tested whether these measurements were different from random expectations.

Results

A mid‐elevation peak in species richness along the elevational gradient was observed, and the lowest richness occurred at the highest elevations. We found two different groups of species, lowland and highland species, with a mixing of groups at intermediate elevations. The turnover component of β‐diversity was significantly higher for both spatial (i.e. elevational) and temporal changes in species composition. However, when the data for genera by site were considered, the elevational turnover value decreased in relative importance. Nestedness‐related processes are more important for temporal dissimilarity patterns at higher elevation sites.

Main conclusions

Spatial and temporal turnover of dung beetle species is the most important component of β‐diversity along the elevational gradient. High‐elevation assemblages are not subsets of assemblages that inhabit lower elevations, but this relationship ceases when β‐diversity is measured at the generic level. Environmental changes across elevations may be the cause of the differential establishment of distinctive species, but these species typically belong to the same higher taxonomic rank. Conservation strategies should consider elevational gradients in case‐specific scenarios as they may contain distinct species assemblages in lowlands vs. highlands.

     

Taxonomic Richness of Yeasts in Japan within Subtropical and Cool Temperate Areas

Background

An understanding of the role of yeasts in the environment has been uncertain because estimates of population size and diversity have often been based on species identifications that were determined from a limited number of phenotypic characteristics. DNA-based species identification has now become widely used, allowing an accurate assessment of species in different habitats. However, there are still problems in classification because some genera are polyphyletic. Consequently, the identification of yeasts and measurement of their diversity at the genus level remains difficult, as does assignment of genera to higher taxonomic ranks.

Methodology/Principal Findings

A total of 1021 yeast strains was isolated from soil samples and plant materials collected from Japan’s subtropical Iriomote Island and the cool temperate Rishiri Island. Based on sequence analyses of the D1/D2 domain of the LSU rRNA gene, these 1021 strains were tentatively classified into 183 species, with apparent new species accounting for approximately half of the total species isolated (60 and 46, Iriomote and Rishiri, respectively). The yeast species composition was statistically different between the two sites with only 15 species in common. Rarefaction curves of respective sources/areas gave distinctive patterns when the threshold of sequence identity became broader, indicating that the yeast diversity was distinct at the different taxonomic levels compared.

Conclusions/Significance

Our isolation study of yeasts in Japan has enabled us to expand the inventory of species diversity because a large number of new species was observed in the sampling areas. Further, we propose use of a particular diversity threshold as an “indicator” to recognize species, genera and higher taxonomic ranks.     

Cryptic species in tropic sands--interactive 3D anatomy, molecular phylogeny and evolution of meiofaunal Pseudunelidae (Gastropoda, Acochlidia)

Background

Towards realistic estimations of the diversity of marine animals, tiny meiofaunal species usually are underrepresented. Since the biological species concept is hardly applicable on exotic and elusive animals, it is even more important to apply a morphospecies concept on the best level of information possible, using accurate and efficient methodology such as 3D modelling from histological sections. Molecular approaches such as sequence analyses may reveal further, cryptic species. This is the first case study on meiofaunal gastropods to test diversity estimations from traditional taxonomy against results from modern microanatomical methodology and molecular systematics.

Results

The examined meiofaunal Pseudunela specimens from several Indo-Pacific islands cannot be distinguished by external features. Their 3D microanatomy shows differences in the organ systems and allows for taxonomic separation in some cases. Additional molecular analyses based on partial mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA markers revealed considerable genetic structure that is largely congruent with anatomical or geographical patterns. Two new species (Pseudunela viatoris and P. marteli spp. nov.) are formally described integrating morphological and genetic analyses. Phylogenetic analysis using partial 16S rRNA, COI and the nuclear 18S rRNA markers shows a clade of Pseudunelidae species as the sister group to limnic Acochlidiidae. Within Pseudunela, two subtypes of complex excretory systems occur. A complex kidney already evolved in the ancestor of Hedylopsacea. Several habitat shifts occurred during hedylopsacean evolution.

Conclusions

Cryptic species occur in tropical meiofaunal Pseudunela gastropods, and likely in other meiofaunal groups with poor dispersal abilities, boosting current diversity estimations. Only a combined 3D microanatomical and molecular approach revealed actual species diversity within Pseudunela reliably. Such integrative methods are recommended for all taxonomic approaches and biodiversity surveys on soft-bodied and small-sized invertebrates. With increasing taxon sampling and details studied, the evolution of acochlidian panpulmonates is even more complex than expected.     

Diversity in a Cold Hot-Spot: DNA-Barcoding Reveals Patterns of Evolution among Antarctic Demosponges (Class Demospongiae,Phylum Porifera)

Background

The approximately 350 demosponge species that have been described from Antarctica represent a faunistic component distinct from that of neighboring regions. Sponges provide structure to the Antarctic benthos and refuge to other invertebrates, and can be dominant in some communities. Despite the importance of sponges in the Antarctic subtidal environment, sponge DNA barcodes are scarce but can provide insight into the evolutionary relationships of this unique biogeographic province.

Methodology/Principal Findings

We sequenced the standard barcoding COI region for a comprehensive selection of sponges collected during expeditions to the Ross Sea region in 2004 and 2008, and produced DNA-barcodes for 53 demosponge species covering about 60% of the species collected. The Antarctic sponge communities are phylogenetically diverse, matching the diversity of well-sampled sponge communities in the Lusitanic and Mediterranean marine provinces in the Temperate Northern Atlantic for which molecular data are readily available. Additionally, DNA-barcoding revealed levels of in situ molecular evolution comparable to those present among Caribbean sponges. DNA-barcoding using the Segregating Sites Algorithm correctly assigned approximately 54% of the barcoded species to the morphologically determined species.

Conclusion/Significance

A barcode library for Antarctic sponges was assembled and used to advance the systematic and evolutionary research of Antarctic sponges. We provide insights on the evolutionary forces shaping Antarctica''s diverse sponge communities, and a barcode library against which future sequence data from other regions or depth strata of Antarctica can be compared. The opportunity for rapid taxonomic identification of sponge collections for ecological research is now at the horizon.     

Coalescent Method in Conjunction with Niche Modeling Reveals Cryptic Diversity among Centipedes in the Western Ghats of South India

Background

There has been growing interest in integrative taxonomy that uses data from multiple disciplines for species delimitation. Typically, in such studies, monophyly is taken as a proxy for taxonomic distinctiveness and these units are treated as potential species. However, monophyly could arise due to stochastic processes. Thus here, we have employed a recently developed tool based on coalescent approach to ascertain the taxonomic distinctiveness of various monophyletic units. Subsequently, the species status of these taxonomic units was further tested using corroborative evidence from morphology and ecology. This inter-disciplinary approach was implemented on endemic centipedes of the genus Digitipes (Attems 1930) from the Western Ghats (WG) biodiversity hotspot of India. The species of the genus Digitipes are morphologically conserved, despite their ancient late Cretaceous origin.

Principal Findings

Our coalescent analysis based on mitochondrial dataset indicated the presence of nine putative species. The integrative approach, which includes nuclear, morphology, and climate datasets supported distinctiveness of eight putative species, of which three represent described species and five were new species. Among the five new species, three were morphologically cryptic species, emphasizing the effectiveness of this approach in discovering cryptic diversity in less explored areas of the tropics like the WG. In addition, species pairs showed variable divergence along the molecular, morphological and climate axes.

Conclusions

A multidisciplinary approach illustrated here is successful in discovering cryptic diversity with an indication that the current estimates of invertebrate species richness for the WG might have been underestimated. Additionally, the importance of measuring multiple secondary properties of species while defining species boundaries was highlighted given variable divergence of each species pair across the disciplines.     

Application of DNA barcoding in biodiversity studies of shallow-water octocorals: molecular proxies agree with morphological estimates of species richness in Palau

The application of DNA barcoding to anthozoan cnidarians has been hindered by their slow rates of mitochondrial gene evolution and the failure to identify alternative molecular markers that distinguish species reliably. Among octocorals, however, multilocus barcodes can distinguish up to 70 % of morphospecies, thereby facilitating the identification of species that are ecologically important but still very poorly known taxonomically. We tested the ability of these imperfect DNA barcodes to estimate species richness in a biodiversity survey of the shallow-water octocoral fauna of Palau using multilocus (COI, mtMutS, 28S rDNA) sequences obtained from 305 specimens representing 38 genera of octocorals. Numbers and identities of species were estimated independently (1) by a taxonomic expert using morphological criteria and (2) by assigning sequences to molecular operational taxonomic units (MOTUs) using predefined genetic distance thresholds. Estimated numbers of MOTUs ranged from 73 to 128 depending on the barcode and distance threshold applied, bracketing the estimated number of 118 morphospecies. Concordance between morphospecies identifications and MOTUs ranged from 71 to 75 % and differed little among barcodes. For the speciose and ecologically dominant genus Sinularia, however, we were able to identify 95 % of specimens correctly simply by comparing mtMutS sequences and in situ photographs of colonies to an existing vouchered database. Because we lack a clear understanding of species boundaries in most of these taxa, numbers of morphospecies and MOTUs are both estimates of the true species diversity, and we cannot currently determine which is more accurate. Our results suggest, however, that the two methods provide comparable estimates of species richness for shallow-water Indo-Pacific octocorals. Use of molecular barcodes in biodiversity surveys will facilitate comparisons of species richness and composition among localities and over time, data that do not currently exist for any octocoral community.     

Elevational Gradients in Fish Diversity in the Himalaya: Water Discharge Is the Key Driver of Distribution Patterns

Background

Studying diversity and distribution patterns of species along elevational gradients and understanding drivers behind these patterns is central to macroecology and conservation biology. A number of studies on biogeographic gradients are available for terrestrial ecosystems, but freshwater ecosystems remain largely neglected. In particular, we know very little about the species richness gradients and their drivers in the Himalaya, a global biodiversity hotspot.

Methodology/Principal Findings

We collated taxonomic and distribution data of fish species from 16 freshwater Himalayan rivers and carried out empirical studies on environmental drivers and fish diversity and distribution in the Teesta river (Eastern Himalaya). We examined patterns of fish species richness along the Himalayan elevational gradients (50–3800 m) and sought to understand the drivers behind the emerging patterns. We used generalized linear models (GLM) and generalized additive models (GAM) to examine the richness patterns; GLM was used to investigate relationship between fish species richness and various environmental variables. Regression modelling involved stepwise procedures, including elimination of collinear variables, best model selection, based on the least Akaike’s information criterion (AIC) and the highest percentage of deviance explained (D²). This maiden study on the Himalayan fishes revealed that total and non-endemic fish species richness monotonously decrease with increasing elevation, while endemics peaked around mid elevations (700–1500 m). The best explanatory model (synthetic model) indicated that water discharge is the best predictor of fish species richness patterns in the Himalayan rivers.

Conclusions/Significance

This study, carried out along one of the longest bioclimatic elevation gradients of the world, lends support to Rapoport’s elevational rule as opposed to mid domain effect hypothesis. We propose a species-discharge model and contradict species-area model in predicting fish species richness. We suggest that drivers of richness gradients in terrestrial and aquatic ecosystems are likely to be different. These studies are crucial in context of the impacts of unprecedented on-going river regulation on fish diversity and distribution in the Himalaya.     

Accurate genome relative abundance estimation for closely related species in a metagenomic sample

Background

Metagenomics has a great potential to discover previously unattainable information about microbial communities. An important prerequisite for such discoveries is to accurately estimate the composition of microbial communities. Most of prevalent homology-based approaches utilize solely the results of an alignment tool such as BLAST, limiting their estimation accuracy to high ranks of the taxonomy tree.

Results

We developed a new homology-based approach called Taxonomic Analysis by Elimination and Correction (TAEC), which utilizes the similarity in the genomic sequence in addition to the result of an alignment tool. The proposed method is comprehensively tested on various simulated benchmark datasets of diverse complexity of microbial structure. Compared with other available methods designed for estimating taxonomic composition at a relatively low taxonomic rank, TAEC demonstrates greater accuracy in quantification of genomes in a given microbial sample. We also applied TAEC on two real metagenomic datasets, oral cavity dataset and Crohn’s disease dataset. Our results, while agreeing with previous findings at higher ranks of the taxonomy tree, provide accurate estimation of taxonomic compositions at the species/strain level, narrowing down which species/strains need more attention in the study of oral cavity and the Crohn’s disease.

Conclusions

By taking account of the similarity in the genomic sequence TAEC outperforms other available tools in estimating taxonomic composition at a very low rank, especially when closely related species/strains exist in a metagenomic sample.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-242) contains supplementary material, which is available to authorized users.     

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.     

Assessing macroinvertebrate biodiversity in freshwater ecosystems: advances and challenges in DNA-based approaches

Assessing the biodiversity of macroinvertebrate fauna in freshwater ecosystems is an essential component of both basic ecological inquiry and applied ecological assessments. Aspects of taxonomic diversity and composition in freshwater communities are widely used to quantify water quality and measure the efficacy of remediation and restoration efforts. The accuracy and precision of biodiversity assessments based on standard morphological identifications are often limited by taxonomic resolution and sample size. Morphologically based identifications are laborious and costly, significantly constraining the sample sizes that can be processed. We suggest that the development of an assay platform based on DNA signatures will increase the precision and ease of quantifying biodiversity in freshwater ecosystems. Advances in this area will be particularly relevant for benthic and planktonic invertebrates, which are often monitored by regulatory agencies. Adopting a genetic assessment platform will alleviate some of the current limitations to biodiversity assessment strategies. We discuss the benefits and challenges associated with DNA-based assessments and the methods that are currently available. As recent advances in microarray and next-generation sequencing technologies will facilitate a transition to DNA-based assessment approaches, future research efforts should focus on methods for data collection, assay platform development, establishing linkages between DNA signatures and well-resolved taxonomies, and bioinformatics.     

Barcoding and border biosecurity: identifying cyprinid fishes in the aquarium trade

Background

Poorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species.

Methodology/Principal Findings

We sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90–99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii.

Conclusions/Significance

We demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade.     

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.