Multi-locus species delimitation in closely related animals and fungi: one marker is not enough

Despite taxonomy’s 250‐year history, the past 20 years have borne witness to remarkable advances in technology and techniques, as well as debate. DNA barcoding has generated a substantial proportion of this debate, with its proposition that a single mitochondrial sequence will consistently identify and delimit species, replacing more evidence‐rich and time‐intensive methods. Although mitochondrial DNA (mtDNA) has since been the focus of voluminous discussion and case studies, little effort has been made to comprehensively evaluate its success in delimiting closely related species. We have conducted the first broadly comparative literature review addressing the efficacy of molecular markers for delimiting such species over a broad taxonomic range. By considering only closely related species, we sought to avoid confusion of success rates with those due to deeply divergent taxa. We also address whether increased population‐level or geographic sampling affects delimitation success. Based on the results from 101 studies, we found that all marker groups had approximately equal success rates (～70%) in delimiting closely related species and that the use of additional loci increased average delimitation success. We also found no relationship between increased sampling of intraspecific variability and delimitation success. Ultimately, our results support a multi‐locus integrative approach to species delimitation and taxonomy.     

Intragenomic variation in nuclear ribosomal markers and its implication in species delimitation,identification and barcoding in fungi

Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.     

DNA Barcoding is not enough: mismatch of taxonomy and genealogy in New Zealand grasshoppers (Orthoptera: Acrididae).

DNA barcoding has been touted as a program that will efficiently and relatively cheaply inform on biological diversity; yet many exemplars purporting to demonstrate the efficacy of the method have been undertaken by its principal proponents. Critics of DNA barcoding identify insufficient within-taxon sampling coupled with the knowledge that levels of haplotypic paraphyly are rather high as key reasons to be sceptical of the value of an exclusively DNA-based taxonomic. Here I applied a DNA barcoding approach using mtDNA sequences from the cytochrome oxidase I gene to examine diversity in a group of endemic New Zealand grasshoppers belonging to the genus Sigaus . The mtDNA data revealed high genetic distances among individuals of a single morpho-species, but this diversity was geographically partitioned. Phylogenetic analysis supported at least four haplogroups within one species ( Sigaus australis ) but paraphyly of this species with respect to several others. In some instances two morphologically and ecologically distinct species shared identical mtDNA haplotypes. The mismatch of genealogy and taxonomy revealed in the Sigaus australis complex indicates that, if used in isolation, DNA barcoding data can be highly misleading about biodiversity. Furthermore, failure to take into account evidence from natural history and morphology when utilizing DNA barcoding will tend to conceal the underlying evolutionary processes associated with speciation.
© The Willi Hennig Society 2007.     

An evaluation of candidate plant DNA barcodes and assignment methods in diagnosing 29 species in the genus Agalinis (Orobanchaceae)

? Premise of the study: DNA barcoding has been proposed as a useful technique within many disciplines (e.g., conservation biology and forensics) for determining the taxonomic identity of a sample based on nucleotide similarity to samples of known taxonomy. Application of DNA barcoding to plants has primarily focused on evaluating the success of candidate barcodes across a broad spectrum of evolutionary divergence. Less attention has been paid to evaluating performance when distinguishing congeners or to differential success of analytical techniques despite the fact that the practical application and utility of barcoding hinges on the ability to distinguish closely related species. ? Methods: We tested the ability to distinguish among 92 samples representing 29 putative species in the genus Agalinis (Orobanchaceae) using 13 candidate barcodes and three analytical methods (i.e., threshold genetic distances, hierarchical tree-based, and diagnostic character differences). Due to questions regarding evolutionary distinctiveness of some taxa, we evaluated success under two taxonomic hypotheses. ? Key results: The psbA-trnH and trnT-trnL barcodes in conjunction with the "best close match" distance-based method best met the objectives of DNA barcoding. Success was also a function of the taxonomy used. ? Conclusions: In addition to accurately identifying query sequences, our results showed that DNA barcoding is useful for detecting taxonomic uncertainty; determining whether erroneous taxonomy or incomplete lineage sorting is the cause requires additional information provided by traditional taxonomic approaches. The magnitude of differentiation within and among the Agalinis species sampled suggests that our results inform how DNA barcoding will perform among closely related species in other genera.     

Mitonuclear discordance in wolf spiders: Genomic evidence for species integrity and introgression

Systematists and taxonomists have benefited greatly from the emergence of molecular methods. Species identification has become straightforward through DNA barcoding and the rapid build‐up of massive DNA barcode reference libraries. In animals, mitonuclear discordance can significantly complicate the process of species identification and delimitation. The causes of mitonuclear discordance are either biological (e.g., introgression, incomplete lineage sorting, horizontal gene transfer androgenesis) or induced by operational factors (e.g., human error with specimen misidentification or incorrect species delimitation). Moreover, endosymbionts may play an important role in promoting fixation of mitochondrial genomes. Here, we study the mitonuclear discordance of wolf spiders species (Lycosidae) (independent cases from Alopecosa aculeata and Pardosa pullata groups) that share identical COI DNA barcodes. We approached the case utilizing double‐digest restriction site‐associated DNA sequencing (ddRADseq) to obtain and analyse genomic‐scale data. Our results suggest that the observed cases of mitonuclear discordance are not due to operational reasons but result from biological processes. Further analysis indicated introgression and that incomplete lineage sorting is unlikely to have been responsible for the observed discrepancy. Additional survey of endosymbionts provided ideas on further research and their role in shaping mitochondrial DNA distribution patterns. Thus, ddRADseq grants an efficient way to study the taxonomy of problematic groups with insight into underlying evolutionary processes.     

Identifying species of moths (Lepidoptera) from Baihua Mountain,Beijing, China,using DNA barcodes

DNA barcoding has become a promising means for the identification of organisms of all life‐history stages. Currently, distance‐based and tree‐based methods are most widely used to define species boundaries and uncover cryptic species. However, there is no universal threshold of genetic distance values that can be used to distinguish taxonomic groups. Alternatively, DNA barcoding can deploy a “character‐based” method, whereby species are identified through the discrete nucleotide substitutions. Our research focuses on the delimitation of moth species using DNA‐barcoding methods. We analyzed 393 Lepidopteran specimens belonging to 80 morphologically recognized species with a standard cytochrome c oxidase subunit I (COI) sequencing approach, and deployed tree‐based, distance‐based, and diagnostic character‐based methods to identify the taxa. The tree‐based method divided the 393 specimens into 79 taxa (species), and the distance‐based method divided them into 84 taxa (species). Although the diagnostic character‐based method found only 39 so‐identifiable species in the 80 species, with a reduction in sample size the accuracy rate substantially improved. For example, in the Arctiidae subset, all 12 species had diagnostics characteristics. Compared with traditional morphological method, molecular taxonomy performed well. All three methods enable the rapid delimitation of species, although they have different characteristics and different strengths. The tree‐based and distance‐based methods can be used for accurate species identification and biodiversity studies in large data sets, while the character‐based method performs well in small data sets and can also be used as the foundation of species‐specific biochips.     

Species limits in molecular phylogenies: a cautionary tale from Australian land snails (Camaenidae: Amplirhagada Iredale, 1933)

By complementing two independent systematic studies published recently on the Western Australian land snail Amplirhagada, we compare levels of morphological variation in shells and genitalia with those in the mitochondrial markers cytochrome c oxidase (COI) and 16S to evaluate the utility of mtDNA markers for delimiting species. We found that penial morphology and mitochondrial divergence are generally highly consistent in delimiting species, while shells have little overall taxonomic utility in these snails. In addition to this qualitative correspondence, there is almost no overlap between intraspecific and interspecific genetic distances in COI, with the highest intraspecific and lowest interspecific distance being 6%. This value is twice the general level suggested as a DNA barcode threshold by some authors and higher than the best average found in stylommatophoran land snails. Although in Amplirhagada land snails DNA barcoding may provide meaningful information as a first‐pass approach towards species delimitation, we argue that this is due only to specific evolutionary circumstances that facilitated a long‐termed separate evolution of mitochondrial lineages along spatial patterns. However, because in general the amounts of morphological and mitochondrial differentiation of species depend on their evolutionary history and age, the mode of speciation, distributional patterns and ecological adaptations, and absence or presence of mechanisms that prevent gene flow across species limits, the applicability of DNA barcoding has to be confirmed by morphological studies for each single group anew. Based on evidence from both molecular and morphological markers, we describe six new species from the Bonaparte Archipelago and revise the taxonomy of a further two. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 337–362.     

Taxonomic challenges in freshwater fishes: a mismatch between morphology and DNA barcoding in fish of the north‐eastern part of the Congo basin

This study evaluates the utility of DNA barcoding to traditional morphology‐based species identifications for the fish fauna of the north‐eastern Congo basin. We compared DNA sequences (COI) of 821 samples from 206 morphologically identified species. Best match, best close match and all species barcoding analyses resulted in a rather low identification success of 87.5%, 84.5% and 64.1%, respectively. The ratio ‘nearest‐neighbour distance/maximum intraspecific divergence’ was lower than 1 for 26.1% of the samples, indicating possible taxonomic problems. In ten genera, belonging to six families, the number of species inferred from mtDNA data exceeded the number of species identified using morphological features; and in four cases indications of possible synonymy were detected. Finally, the DNA barcodes confirmed previously known identification problems within certain genera of the Clariidae, Cyprinidae and Mormyridae. Our results underscore the large number of taxonomic problems lingering in the taxonomy of the fish fauna of the Congo basin and illustrate why DNA barcodes will contribute to future efforts to compile a reliable taxonomic inventory of the Congo basin fish fauna. Therefore, the obtained barcodes were deposited in the reference barcode library of the Barcode of Life Initiative.     

Genetic Polymorphism of 11 Allozyme Loci in Populations of Wall Lizards (Podarcis sp.) from the Iberian Peninsula and North Africa

The taxonomy of Iberian and North African wall lizards (Podarcis sp.) has been controversial. Recently, morphological and mtDNA sequence data have provided new information on differentiation within these lacertids. To compare these results to those provided by nuclear markers, we investigated variation at 11 polymorphic protein loci using conventional electrophoresis and isoelectric focusing in 11 populations belonging to seven different mtDNA lineages. A total of 62 alleles were found. Populations belonging to the same mtDNA type presented high genetic similarity, whereas strong differentiation was observed between groups. These results are consistent with those previously obtained from morphological and mtDNA analysis and support the idea that Iberian and North African Podarcis are composed of several well-differentiated entities, some of which are already recognized as species, whereas others (belonging to the P. hispanica complex) clearly need taxonomic revision.     

Updated checklist and DNA barcode‐based species delimitations reveal taxonomic uncertainties among freshwater fishes from the mid‐north‐eastern Caatinga ecoregion,north‐eastern Brazil

The mid‐north‐eastern Caatinga is a semiarid freshwater ecoregion in North‐eastern Brazil that is dominated by temporary rivers and is currently classified as one of the least ichthyologically‐known ecoregions in the world. The present study aimed to provide an updated checklist of mid‐north‐eastern Caatinga ecoregion (MNCE) freshwater fish species and evaluate their taxonomic identity using morphology, DNA barcoding and multiple species delimitation approaches. After reviewing published studies and ichthyological collections, 119 species were identified. Among these were 94 putatively valid native and 14 non‐native species, five undescribed native species, four new records for the MNCE, 11 potential cases of misidentification and 14 species listed as inquirenda. Additionally, 252 individuals from 49 species were barcoded, revealing three potential taxonomic synonyms. The combined molecular approaches estimated a total of 91 native species, although a finalized species list for the MNCE awaits additional taxonomic revisions and field surveys. This study provides the most up‐to‐date species checklist for the MNCE and a molecular reference database for identifying MNCE fishes with DNA barcodes. Results highlight the need to integrate traditional taxonomy with molecular approaches to correctly identify species, especially in taxonomically problematic ecoregions such as the MNCE.     

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost‐effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non‐trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost‐effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single‐locus DNA barcode for plants.     

The unholy trinity: taxonomy, species delimitation and DNA barcoding

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this 'DNA barcoding' initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the 'DNA barcoding' initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the 'DNA barcoding' initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings--Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of 'DNA barcoding'.     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

Molecular operational taxonomic units as approximations of species in the light of evolutionary models and empirical data from Fungi

During the last couple of decades, an increasing number of studies use sequence clusters as units for taxonomic diversity. It is well known that such molecular operational taxonomic units (MOTUs) do not necessarily correspond to species, but they are treated as such when measuring diversity and testing theories. Here, I show that data from studies of molecular evolution and species diversification of fungi indicate that commonly used cut‐offs are likely to lump species in many cases. At the same time, empirical studies show that the mean within‐species variation is close to these cut‐offs. That the within‐species variation estimates are plausible is supported by coalescence modelling under a range of parameter settings. In addition, studies using crossing tests to delimit species show that there often is an overlap in within‐ and between‐species distances. The available data therefore indicate that sequence clusters are likely to misrepresent species. However, to keep a biological relevance, MOTUs should be kept in close agreement with species. Studies using them should therefore asses how sensitive the results are to differences between MOTUs and species – something that is rarely done. An even better solution is to directly include the uncertainty in species delimitation in the analyses, but in many cases, we need to increase our knowledge of taxonomy and evolution to do this accurately. Even if the empirical data referred to here pertain to the “barcoding” region of rDNA in fungi, there is nothing indicating that the situation is substantially better for other taxa or genes.     

Species delimitation and digit number in a North African skink

Delimitation of species is an important and controversial area within evolutionary biology. Many species boundaries have been defined using morphological data. New genetic approaches now offer more objective evaluation and assessment of the reliability of morphological variation as an indicator that speciation has occurred. We examined geographic variation in morphology of the continuously distributed skink Chalcides mionecton from Morocco and used Bayesian analyses of nuclear and mitochondrial DNA (mtDNA) loci to examine: (i) their concordance with morphological patterns, (ii) support for species delimitation, (iii) timing of speciation, and (iv) levels of gene flow between species. Four digit individuals were found at sites between Cap Rhir (in the south) and the northern extreme of the range, whereas five‐digit individuals were found in two disjunct areas: (i) south of Cap Rhir and (ii) the north of the range where they were often syntopic with four‐digit individuals. The pattern of variation in generalized body dimensions was largely concordant with that in digit number, suggesting two general morphotypes. Bayesian analyses of population structure showed that individuals from sites south of Cap Rhir formed one genetic cluster, but that northern four‐ and five‐digit individuals clustered together. Statistical support for delimitation of these genetic clusters into two species was provided by a recent Bayesian method. Phylogenetic–coalescent dating with external time calibrations indicates that speciation was relatively recent, with a 95% posterior interval of 0.46–2.66 mya. This postdates equivalent phylogenetic dating estimates of sequence divergence by approximately 1 Ma. Statistical analyses of a small number of independent loci provide important insights into the history of the speciation process in C. mionecton and support delimitation of populations into two species with distributions that are spatially discordant with patterns of morphological variation.     

Genome‐wide single‐nucleotide polymorphism data reveal cryptic species within cryptic freshwater snail species—The case of the Ancylus fluviatilis species complex

DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis, for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A–C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high‐resolution genome‐wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome‐wide data for species delimitation in such groups.     

DNA条形码分析方法研究进展

DNA条形码是一段短的、标准化的DNA序列,DNA条形码技术通过对DNA条形码序列分析实现物种的有效鉴定.随着生物DNA条形码序列的大量测定,DNA条形码分析方法得到迅速发展,推动了其在生物分子鉴定中的应用.2003年以来,DNA条形码技术已广泛应用于动物、植物和真菌等物种的鉴定,并有力地推动了生物分类学、生物多样性和生态学等学科的发展.本文在综述DNA条形码技术的基础上,总结了5类主要的DNA条形码分析方法,即基于遗传距离的分析、基于遗传相似度的分析、基于系统发育树的分析、基于序列特征的分析和基于统计分类法的分析,并进一步展望了DNA条形码技术的发展与应用.     

红菇科可食真菌的若干分类问题

红菇科Russulaceae包含大量全球广泛采食的野生食用菌,同时也有一定数目的毒菌。该科特别是红菇属的分类是大型真菌分类的难点。近年来DNA数据大量应用于红菇科的分类,更新了属的界定和概念,发现了大量新物种,为食用菌和毒菌的识别和鉴定带来了可用的名称。然而,DNA证据并不总是与形态证据吻合,这又为食用菌和毒菌的识别和名称的使用带来了困扰和不便。本文针对乳菇属、多汁乳菇属和红菇属中的重要食用菌类群,回顾了近年来的分类研究进展,分析了研究背后的数据实情和存在的分类问题。认为：在食用菌和毒菌的确定上,依靠物种复合群共有的形态特征更具有可操作性;依据DNA序列进行的劈分式分类和依靠少数样品的特征及DNA序列上的少量差异发表新种的做法可能产生不便于使用的后果;在乳菇属和红菇属中,“BLAST相似度低的即为新种”的分类实践存在错误风险;充分结合历史资料和各个类群的特点,确定物种划分的阈值,才能有望解决红菇科真菌的分类问题。     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.