Single nucleotide polymorphism barcoding of cytochrome c oxidase I sequences for discriminating 17 species of Columbidae by decision tree algorithm

DNA barcodes are widely used in taxonomy, systematics, species identification, food safety, and forensic science. Most of the conventional DNA barcode sequences contain the whole information of a given barcoding gene. Most of the sequence information does not vary and is uninformative for a given group of taxa within a monophylum. We suggest here a method that reduces the amount of noninformative nucleotides in a given barcoding sequence of a major taxon, like the prokaryotes, or eukaryotic animals, plants, or fungi. The actual differences in genetic sequences, called single nucleotide polymorphism (SNP) genotyping, provide a tool for developing a rapid, reliable, and high‐throughput assay for the discrimination between known species. Here, we investigated SNPs as robust markers of genetic variation for identifying different pigeon species based on available cytochrome c oxidase I (COI) data. We propose here a decision tree‐based SNP barcoding (DTSB) algorithm where SNP patterns are selected from the DNA barcoding sequence of several evolutionarily related species in order to identify a single species with pigeons as an example. This approach can make use of any established barcoding system. We here firstly used as an example the mitochondrial gene COI information of 17 pigeon species (Columbidae, Aves) using DTSB after sequence trimming and alignment. SNPs were chosen which followed the rule of decision tree and species‐specific SNP barcodes. The shortest barcode of about 11 bp was then generated for discriminating 17 pigeon species using the DTSB method. This method provides a sequence alignment and tree decision approach to parsimoniously assign a unique and shortest SNP barcode for any known species of a chosen monophyletic taxon where a barcoding sequence is available.     

Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan

Background

DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking.

Methodology/Principal Findings

The Japanese pteridophyte flora (733 taxa including subspecies and varieties) was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA) with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0%) taxa for rbcL and 617 (84.2%) taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances) did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52%) was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only.

Conclusions/Significance

This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially anticipated to be helpful for identification of non-hybridizing gametophytes.     

Polyhedral Geometry of Phylogenetic Rogue Taxa

It is well known among phylogeneticists that adding an extra taxon (e.g. species) to a data set can alter the structure of the optimal phylogenetic tree in surprising ways. However, little is known about this “rogue taxon” effect. In this paper we characterize the behavior of balanced minimum evolution (BME) phylogenetics on data sets of this type using tools from polyhedral geometry. First we show that for any distance matrix there exist distances to a “rogue taxon” such that the BME-optimal tree for the data set with the new taxon does not contain any nontrivial splits (bipartitions) of the optimal tree for the original data. Second, we prove a theorem which restricts the topology of BME-optimal trees for data sets of this type, thus showing that a rogue taxon cannot have an arbitrary effect on the optimal tree. Third, we computationally construct polyhedral cones that give complete answers for BME rogue taxon behavior when our original data fits a tree on four, five, and six taxa. We use these cones to derive sufficient conditions for rogue taxon behavior for four taxa, and to understand the frequency of the rogue taxon effect via simulation.     

Barcoding diatoms: evaluation of the V4 subregion on the 18S rRNA gene,including new primers and protocols

Diatoms are present in all types of water bodies and their species diversity is influenced greatly by environmental conditions. This means that diatom occurrence and abundances are suitable indicators of water quality. Furthermore, continuous screening of algal biodiversity can provide information about diversity changes in ecosystems. Thus, diatoms represent a desirable group for which to develop an easy to use, quick, efficient, and standardised organism identification tool to serve routine water quality assessments. Because conventional morphological identification of diatoms demands specialised in-depth knowledge, we have established standard laboratory procedures for DNA barcoding in diatoms. We (1) identified a short segment (about 400 bp) of the SSU (18S) rRNA gene which is applicable for the identification of diatom taxa, and (2) elaborated a routine protocol including standard primers for this group of microalgae. To test the universality of the primer binding sites and the discriminatory power of the proposed barcode region, 123 taxa, representing limnic diatom diversity, were included in the study and identified at species level. The effectiveness of the barcode was also scrutinised within a closely related species group, namely the Sellaphora pupula taxon complex and relatives.     

High mitochondrial diversity in geographically widespread butterflies of Madagascar: A test of the DNA barcoding approach

The standardized use of mitochondrial cytochrome c oxidase subunit I (COI) gene sequences as DNA barcodes has been widely promoted as a high-throughput method for species identification and discovery. Species delimitation has been based on the following criteria: (1) monophyletic association and less frequently (2) a minimum 10× greater divergence between than within species. Divergence estimates, however, can be inflated if sister species pairs are not included and the geographic extent of variation within any given taxon is not sampled comprehensively. This paper addresses both potential biases in DNA divergence estimation by sampling range-wide variation in several morphologically distinct, endemic butterfly species in the genus Heteropsis, some of which are sister taxa. We also explored the extent to which mitochondrial DNA from the barcode region can be used to assess the effects of historical rainforest fragmentation by comparing genetic variation across Heteropsis populations with an unrelated forest-associated taxon Saribia tepahi. Unexpectedly, generalized primers led to the inadvertent amplification of the endosymbiont Wolbachia, undermining the use of universal primers and necessitating the design of genus-specific COI primers alongside a Wolbachia-specific PCR assay. Regardless of the high intra-specific genetic variation observed, most species satisfy DNA barcoding criteria and can be differentiated in the nuclear phylogeny. Nevertheless, two morphologically distinguishable candidate species fail to satisfy the barcoding 10× genetic distance criterion, underlining the difficulties of applying a standard distance threshold to species delimitation. Phylogeographic analysis of COI data suggests that forest fragmentation may have played an important role in the recent evolutionary diversification of these butterflies. Further work on other Malagasy taxa using both mitochondrial and nuclear data will provide better insight into the role of historical habitat fragmentation in species diversification and may potentially contribute to the identification of priority areas for conservation.     

Incomplete estimates of genetic diversity within species: Implications for DNA barcoding

DNA barcoding has greatly accelerated the pace of specimen identification to the species level, as well as species delineation. Whereas the application of DNA barcoding to the matching of unknown specimens to known species is straightforward, its use for species delimitation is more controversial, as species discovery hinges critically on present levels of haplotype diversity, as well as patterning of standing genetic variation that exists within and between species. Typical sample sizes for molecular biodiversity assessment using DNA barcodes range from 5 to 10 individuals per species. However, required levels that are necessary to fully gauge haplotype variation at the species level are presumed to be strongly taxon‐specific. Importantly, little attention has been paid to determining appropriate specimen sample sizes that are necessary to reveal the majority of intraspecific haplotype variation within any one species. In this paper, we present a brief outline of the current literature and methods on intraspecific sample size estimation for the assessment of COI DNA barcode haplotype sampling completeness. The importance of adequate sample sizes for studies of molecular biodiversity is stressed, with application to a variety of metazoan taxa, through reviewing foundational statistical and population genetic models, with specific application to ray‐finned fishes (Chordata: Actinopterygii). Finally, promising avenues for further research in this area are highlighted.     

Minimizing polymerase biases in metabarcoding

DNA metabarcoding is an increasingly popular method to characterize and quantify biodiversity in environmental samples. Metabarcoding approaches simultaneously amplify a short, variable genomic region, or “barcode,” from a broad taxonomic group via the polymerase chain reaction (PCR), using universal primers that anneal to flanking conserved regions. Results of these experiments are reported as occurrence data, which provide a list of taxa amplified from the sample, or relative abundance data, which measure the relative contribution of each taxon to the overall composition of amplified product. The accuracy of both occurrence and relative abundance estimates can be affected by a variety of biological and technical biases. For example, taxa with larger biomass may be better represented in environmental samples than those with smaller biomass. Here, we explore how polymerase choice, a potential source of technical bias, might influence results in metabarcoding experiments. We compared potential biases of six commercially available polymerases using a combination of mixtures of amplifiable synthetic sequences and real sedimentary DNA extracts. We find that polymerase choice can affect both occurrence and relative abundance estimates and that the main source of this bias appears to be polymerase preference for sequences with specific GC contents. We further recommend an experimental approach for metabarcoding based on results of our synthetic experiments.     

Design of character‐based DNA barcode motif for species identification: A computational approach and its validation in fishes

The DNA barcodes are generally interpreted using distance‐based and character‐based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance‐based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character‐based approach more accurately defines this using a unique set of nucleotide characters. The character‐based analysis of full‐length barcode has some inherent limitations, like sequencing of the full‐length barcode, use of a sparse‐data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154‐bp fragment, from the transversion‐rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species‐specific barcode motifs for 109 species by the character‐based method, which successfully identifies the correct species using a pattern‐matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species‐specific mini‐barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini‐barcode approach will greatly benefit the field‐based system of rapid species identification.     

Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini‐barcode

Bees (Apidae), of which there are more than 19 900 species, are extremely important for ecosystem services and economic purposes, so taxon identity is a major concern. The goal of this study was to optimize the DNA barcode technique based on the Cytochrome c oxidase (COI) mitochondrial gene region. This approach has previously been shown to be useful in resolving taxonomic inconsistencies and for species identification when morphological data are poor. Specifically, we designed and tested new primers and standardized PCR conditions to amplify the barcode region for bees, focusing on the corbiculate Apids. In addition, primers were designed to amplify small COI amplicons and tested with pinned specimens. Short barcode sequences were easily obtained for some Bombus century‐old museum specimens and shown to be useful as mini‐barcodes. The new primers and PCR conditions established in this study proved to be successful for the amplification of the barcode region for all species tested, regardless of the conditions of tissue preservation. We saw no evidence of Wolbachia or numts amplification by these primers, and so we suggest that these new primers are of broad value for corbiculate bee identification through DNA barcode.     

Phylogeny of the Lemuridae: Effects of Character and Taxon Sampling on Resolution of Species Relationships within Eulemur

DNA sequences from three mitochondrial genes and one nuclear gene were analyzed to determine the phylogeny of the Malagasy primate family Lemuridae. Whether analyzed separately or in combination, the data consistently indicate that Eulemur species comprise a clade that is sister to a Lemur catta plus Hapalemur clade. The genus Varecia is basal to both. Resolution of cladogenic events within Eulemur was found to be extremely problematic with a total of six alternative arrangements offered by various data sets and weighting regimes. We attempt to determine the best arrangement of Eulemur taxa through a variety of character and taxon sampling strategies. Because our study includes all but one Eulemur species, increased taxon sampling is probably not an option for enhancing phylogenetic accuracy. We find, however, that the combined genetic data set is more robust to changes in taxon sample than are any of the individual data sets, suggesting that increased character sampling stabilizes phylogenetic resolution. Nonetheless, due to the difficult nature of the problem, we may have to accept certain aspects of Eulemur interrelationships as uncertain.     

Can Global Weed Assemblages Be Used to Predict Future Weeds?

Predicting which plant taxa are more likely to become weeds in a region presents significant challenges to both researchers and government agencies. Often it is done in a qualitative or semi-quantitative way. In this study, we explored the potential of using the quantitative self-organising map (SOM) approach to analyse global weed assemblages and estimate likelihoods of plant taxa becoming weeds before and after they have been moved to a new region. The SOM approach examines plant taxa associations by analysing where a taxon is recorded as a weed and what other taxa are recorded as weeds in those regions. The dataset analysed was extracted from a pre-existing, extensive worldwide database of plant taxa recorded as weeds or other related status and, following reformatting, included 187 regions and 6690 plant taxa. To assess the value of the SOM approach we selected Australia as a case study. We found that the key and most important limitation in using such analytical approach lies with the dataset used. The classification of a taxon as a weed in the literature is not often based on actual data that document the economic, environmental and/or social impact of the taxon, but mostly based on human perceptions that the taxon is troublesome or simply not wanted in a particular situation. The adoption of consistent and objective criteria that incorporate a standardized approach for impact assessment of plant taxa will be necessary to develop a new global database suitable to make predictions regarding weediness using methods like SOM. It may however, be more realistic to opt for a classification system that focuses on the invasive characteristics of plant taxa without any inference to impacts, which to be defined would require some level of research to avoid bias from human perceptions and value systems.     

Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina)

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (~450 bp) representing ~100 morphospecies from ~650 collections of Agaricomycotina using several sets of new primers. Large introns (~1500 bp) at variable locations were detected in ~5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (~30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.     

利用植物DNA条形码构建亚热带森林群落系统发育关系——以鼎湖山样地为例

在群落水平上重建植物系统发育关系是当前植物系统学研究的一项重要内容;DNA条形码技术的出现为这一工作的开展提供了便利。本文选取国际通用的植物DNA条形码（rbcL,matK和psbA trnH）,对鼎湖山大样地的183个物种（隶属于24目51科110属）进行测序;分别利用两位点和三位点DNA条形码组合构建该样地植物群落的系统发育关系,并比较不同位点组合构建出的群落系统发育关系的拓扑结构和节点支持率;最后选出一个具有最好拓扑结构和最高节点支持率的鼎湖山大样地群落系统发育关系。在目、科和属这三个水平上,三位点条形码片段组合构建的群落系统发育关系与APG系统获得较好匹配;有些进化分支在相应的APG系统位置解决得不好,却在条形码序列构建的系统发育关系中得到了较好解决。表明综合使用不同进化速率的DNA条形码片段并采取三位点超级矩阵的组合策略,在未采用APG系统大框架的情况下,也能快速而又相对准确地构建出鼎湖山南亚热带森林植物群落的系统发育关系。     

DNA barcoding should accompany taxonomy - the case of Cerebratulus spp (Nemertea)

Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus.     

Integrating a comprehensive DNA barcode reference library with a global map of yews (Taxus L.) for forensic identification

Rapid and accurate identification of endangered species is a critical component of biosurveillance and conservation management, and potentially policing illegal trades. However, this is often not possible using traditional taxonomy, especially where only small or preprocessed parts of plants are available. Reliable identification can be achieved via a comprehensive DNA barcode reference library, accompanied by precise distribution data. However, these require extensive sampling at spatial and taxonomic scales, which has rarely been achieved for cosmopolitan taxa. Here, we construct a comprehensive DNA barcode reference library and generate distribution maps using species distribution modelling (SDM), for all 15 Taxus species worldwide. We find that trnL‐trnF is the ideal barcode for Taxus: It can distinguish all Taxus species and in combination with ITS identify hybrids. Among five analysis methods tested, NJ was the most effective. Among 4,151 individuals screened for trnL‐trnF, 73 haplotypes were detected, all species‐specific and some population private. Taxonomical, geographical and genetic dimensions of sampling strategy were all found to affect the comprehensiveness of the resulting DNA barcode library. Maps from SDM showed that most species had allopatric distributions, except T. mairei in the Sino‐Himalayan region. Using the barcode library and distribution map data, two unknown forensic samples were identified to species (and in one case, population) level and another was determined as a putative interspecific hybrid. This integrated species identification system for Taxus can be used for biosurveillance, conservation management and to monitor and prosecute illegal trade. Similar identification systems are recommended for other IUCN‐ and CITES‐listed taxa.     

A DNA barcode library for Korean Chironomidae (Insecta: Diptera) and indexes for defining barcode gap

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis’ sequences to the library.     

利用植物DNA条形码构建亚热带森林群落系统发育关系——以鼎湖山样地为例

在群落水平上重建植物系统发育关系是当前植物系统学研究的一项重要内容;DNA条形码技术的出现为这一工作的开展提供了便利。本文选取国际通用的植物DNA条形码（rbcL,matK和psbA trnH）,对鼎湖山大样地的183个物种（隶属于24目51科110属）进行测序;分别利用两位点和三位点DNA条形码组合构建该样地植物群落的系统发育关系,并比较不同位点组合构建出的群落系统发育关系的拓扑结构和节点支持率;最后选出一个具有最好拓扑结构和最高节点支持率的鼎湖山大样地群落系统发育关系。在目、科和属这三个水平上,三位点条形码片段组合构建的群落系统发育关系与APG系统获得较好匹配;有些进化分支在相应的APG系统位置解决得不好,却在条形码序列构建的系统发育关系中得到了较好解决。表明综合使用不同进化速率的DNA条形码片段并采取三位点超级矩阵的组合策略,在未采用APG系统大框架的情况下,也能快速而又相对准确地构建出鼎湖山南亚热带森林植物群落的系统发育关系。     

Assessing the value of DNA barcodes for molecular phylogenetics: effect of increased taxon sampling in lepidoptera

Background

A common perception is that DNA barcode datamatrices have limited phylogenetic signal due to the small number of characters available per taxon. However, another school of thought suggests that the massively increased taxon sampling afforded through the use of DNA barcodes may considerably increase the phylogenetic signal present in a datamatrix. Here I test this hypothesis using a large dataset of macrolepidopteran DNA barcodes.

Methodology/Principal Findings

Taxon sampling was systematically increased in datamatrices containing macrolepidopteran DNA barcodes. Sixteen family groups were designated as concordance groups and two quantitative measures; the taxon consistency index and the taxon retention index, were used to assess any changes in phylogenetic signal as a result of the increase in taxon sampling. DNA barcodes alone, even with maximal taxon sampling (500 species per family), were not sufficient to reconstruct monophyly of families and increased taxon sampling generally increased the number of clades formed per family. However, the scores indicated a similar level of taxon retention (species from a family clustering together) in the cladograms as the number of species included in the datamatrix was increased, suggesting substantial phylogenetic signal below the ‘family’ branch.

Conclusions/Significance

The development of supermatrix, supertree or constrained tree approaches could enable the exploitation of the massive taxon sampling afforded through DNA barcodes for phylogenetics, connecting the twigs resolved by barcodes to the deep branches resolved through phylogenomics.     

EST-SSRs as a resource for population genetic analyses

Simple-sequence repeats (SSRs) have increasingly become the marker of choice for population genetic analyses. Unfortunately, the development of traditional 'anonymous' SSRs from genomic DNA is costly and time-consuming. These problems are further compounded by a paucity of resources in taxa that lack clear economic importance. However, the advent of the genomics age has resulted in the production of vast amounts of publicly available DNA sequence data, including large collections of expressed sequence tags (ESTs) from a variety of different taxa. Recent research has revealed that ESTs are a potentially rich source of SSRs that reveal polymorphisms not only within the source taxon, but in related taxa, as well. In this paper, we review what is known about the transferability of EST-SSRs from one taxon to another with particular reference to the potential of these markers to facilitate population genetic studies. As an example of the utility of these resources, we then cross-reference existing EST databases against lists of rare, endangered and invasive plant species and conclude that half of all suitable EST databases could be exploited for the population genetic analysis of species of conservation concern. We then discuss the advantages and disadvantages of EST-SSRs in the context of population genetic applications.