metaxa2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data

The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species‐ or genus‐level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The metaxa software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to metaxa – metaxa 2 – that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short‐read (100 bp) and paired‐end sequences, among other changes. The performance of metaxa 2 was compared to other commonly used taxonomic classifiers, showing that metaxa 2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. metaxa 2 is freely available from http://microbiology.se/software/metaxa2/ .     

The nature and status of Lysimachia X producta (Primulaceae)—II

Data from chromosome numbers and from morphological variation in wild plants and artificial hybrids further support the hypothesis thatLysimachia Xproducta is of hybrid origin and the taxonomic judgment that it is best treated at the status of interspecific hybrid rather than as a species of hybrid origin. Additional distribution data are presented.     

Deciphering the products of evolution at the species level: the need for an integrative taxonomy

Progress in molecular techniques together with the incorporation of phylogenetic analyses of DNA into taxonomy have caused an increase in the number of species’ discoveries in groups with morphological characters that are difficult to study or in those containing polytypic species. But some emerged criticisms plead for a taxonomic conservatism grounded either on the requirement of providing evidences of morphological distinctiveness or reproductive barriers to erect new species names. In a case study of taxonomic research on Neotropical frogs, we combine several lines of evidence (morphological characters, prezygotic reproductive isolation and phylogenetic analyses of mitochondrial DNA) to test the status of 15 nominal species and to assess the degree of agreement of the different lines of evidence. Our study reveals that morphology alone is not sufficient to uncover all species, as there is no other single line of evidence independently. Full congruence between lines of evidence is restricted to only four out of the 15 species. Five species show congruence of two lines of evidence, whereas the remaining six are supported by only one. The use of divergence in morphological characters seems to be the most conservative approach to delineate species boundaries because it does not allow the identification of some sibling reciprocally monophyletic species differing in their advertisement calls. The separate analysis of differences in advertisement calls (evidence of reproductive isolation) or of phylogenetic data alone also shows limitations, because they do not support some morphological species. Our study shows that only an integrative approach combining all sources of evidence provides the necessary feedback to evaluate the taxonomic status of existing species and to detect putative new ones. Furthermore, the application of integrative taxonomy enables the identification of hypotheses about the existence of species that will probably be rejected or changed, and those that can be expected to persist.     

新版DELTA系统在植物分类学中的应用——以羊茅属研究为例

DELTA系统是针对分类学研究开发的综合性多功能软件包。新版DELTA系统在运行环境、程序、指令、界面、操作方式及中文字符处理等重要功能上比旧版本均有较大改进。作为国际分类学数据库数据交换标准,应用该系统可以规范分类学描述,对研究中所有常用性状进行识别和处理,自动生成各个研究对象分类学特征描述并对其进行详尽的相似性和差异性比较,对性状特征和分类单元进行注释和图像示例,将数据转换为数量分类学或分支分类学所需格式以生成树状图,自动生成检索表,建成基于本地和网络的交互式专家辅助鉴定系统等。DELTA系统作为植物分类学研究的常用手段和工具是非常适合及大有帮助的。     

Integration of morphological data sets for phylogenetic analysis of Amniota: the importance of integumentary characters and increased taxonomic sampling

Several mutually exclusive hypotheses have been advanced to explain the phylogenetic position of turtles among amniotes. Traditional morphology-based analyses place turtles among extinct anapsids (reptiles with a solid skull roof), whereas more recent studies of both morphological and molecular data support an origin of turtles from within Diapsida (reptiles with a doubly fenestrated skull roof). Evaluation of these conflicting hypotheses has been hampered by nonoverlapping taxonomic samples and the exclusion of significant taxa from published analyses. Furthermore, although data from soft tissues and anatomical systems such as the integument may be particularly relevant to this problem, they are often excluded from large-scale analyses of morphological systematics. Here, conflicting hypotheses of turtle relationships are tested by (1) combining published data into a supermatrix of morphological characters to address issues of character conflict and missing data; (2) increasing taxonomic sampling by more than doubling the number of operational taxonomic units to test internal relationships within suprageneric ingroup taxa; and (3) increasing character sampling by approximately 25% by adding new data on the osteology and histology of the integument, an anatomical system that has been historically underrepresented in morphological systematics. The morphological data set assembled here represents the largest yet compiled for Amniota. Reevaluation of character data from prior studies of amniote phylogeny favors the hypothesis that turtles indeed have diapsid affinities. Addition of new ingroup taxa alone leads to a decrease in overall phylogenetic resolution, indicating that existing characters used for amniote phylogeny are insufficient to explain the evolution of more highly nested taxa. Incorporation of new data from the soft and osseous components of the integument, however, helps resolve relationships among both basal and highly nested amniote taxa. Analysis of a data set compiled from published sources and data original to this study supports monophyly of Amniota, Synapsida, Reptilia, Parareptilia, Eureptilia, Eosuchia, Diapsida, Neodiapsida, Sauria, Lepidosauria, and Archosauriformes, as well as several more highly nested divisions within the latter two clades. Turtles are here resolved as the sister taxon to a monophyletic Lepidosauria (squamates + Sphenodon), a novel phylogenetic position that nevertheless is consistent with recent molecular and morphological studies that have hypothesized diapsid affinities for this clade.     

Taxonomic sufficiency in detecting hydrological changes and reproducing ordination patterns: A test using planktonic ciliates

Ciliates are commonly and successfully used as bioindicators in marine ecosystems, even at low levels of taxonomic resolution. However, the use of these organisms in biomonitoring programs of freshwater ecosystems is less common. Evidence showing the reliability of the taxonomic sufficiency approach for freshwater ciliates is also limited. Demonstrating taxonomic sufficiency would be important to foster the use of ciliates in freshwater biomonitoring programs in a cost-effective way. Here we tested whether richness and multivariate patterns generated by ciliate community data, at species level, were retrieved by analyses carried out with data at lower taxonomic resolutions. Community and environmental datasets were gathered at 36 sites in the Upper Paraná River floodplain (Brazil), during high and low water levels periods of the years 2010 and 2011. We found that, in comparison with species-level data, genus-level identification was sufficient to detect the effects of the environmental changes caused by floods, to retrieve the ordination patterns generated during each hydrological period and to detect relationships with environmental and spatial gradients. We also showed that the use of coarser taxonomic resolutions was not advisable because high relationships with species-level data were found only with genus-level data. We encourage the use of ciliates in biomonitoring programs of freshwater ecosystems and, according to our results, the use of genus-level identification is a reliable strategy, not only to increase cost-efficiency, but also to guarantee temporal comparability of data.     

Systematic Identification of Gene Families for Use as “Markers” for Phylogenetic and Phylogeny-Driven Ecological Studies of Bacteria and Archaea and Their Major Subgroups

With the astonishing rate that genomic and metagenomic sequence data sets are accumulating, there are many reasons to constrain the data analyses. One approach to such constrained analyses is to focus on select subsets of gene families that are particularly well suited for the tasks at hand. Such gene families have generally been referred to as “marker” genes. We are particularly interested in identifying and using such marker genes for phylogenetic and phylogeny-driven ecological studies of microbes and their communities (e.g., construction of species trees, phylogenetic based assignment of metagenomic sequence reads to taxonomic groups, phylogeny-based assessment of alpha- and beta-diversity of microbial communities from metagenomic data). We therefore refer to these as PhyEco (for phylogenetic and phylogenetic ecology) markers. The dual use of these PhyEco markers means that we needed to develop and apply a set of somewhat novel criteria for identification of the best candidates for such markers. The criteria we focused on included universality across the taxa of interest, ability to be used to produce robust phylogenetic trees that reflect as much as possible the evolution of the species from which the genes come, and low variation in copy number across taxa.We describe here an automated protocol for identifying potential PhyEco markers from a set of complete genome sequences. The protocol combines rapid searching, clustering and phylogenetic tree building algorithms to generate protein families that meet the criteria listed above. We report here the identification of PhyEco markers for different taxonomic levels including 40 for “all bacteria and archaea”, 114 for “all bacteria (greatly expanding on the ∼30 commonly used), and 100 s to 1000 s for some of the individual phyla of bacteria. This new list of PhyEco markers should allow much more detailed automated phylogenetic and phylogenetic ecology analyses of these groups than possible previously.     

Outlier detection in BLAST hits

Background

An important task in a metagenomic analysis is the assignment of taxonomic labels to sequences in a sample. Most widely used methods for taxonomy assignment compare a sequence in the sample to a database of known sequences. Many approaches use the best BLAST hit(s) to assign the taxonomic label. However, it is known that the best BLAST hit may not always correspond to the best taxonomic match. An alternative approach involves phylogenetic methods, which take into account alignments and a model of evolution in order to more accurately define the taxonomic origin of sequences. Similarity-search based methods typically run faster than phylogenetic methods and work well when the organisms in the sample are well represented in the database. In contrast, phylogenetic methods have the capability to identify new organisms in a sample but are computationally quite expensive.

Results

We propose a two-step approach for metagenomic taxon identification; i.e., use a rapid method that accurately classifies sequences using a reference database (this is a filtering step) and then use a more complex phylogenetic method for the sequences that were unclassified in the previous step. In this work, we explore whether and when using top BLAST hit(s) yields a correct taxonomic label. We develop a method to detect outliers among BLAST hits in order to separate the phylogenetically most closely related matches from matches to sequences from more distantly related organisms. We used modified BILD (Bayesian Integral Log-Odds) scores, a multiple-alignment scoring function, to define the outliers within a subset of top BLAST hits and assign taxonomic labels. We compared the accuracy of our method to the RDP classifier and show that our method yields fewer misclassifications while properly classifying organisms that are not present in the database. Finally, we evaluated the use of our method as a pre-processing step before more expensive phylogenetic analyses (in our case TIPP) in the context of real 16S rRNA datasets.

Conclusion

Our experiments make a good case for using a two-step approach for accurate taxonomic assignment. We show that our method can be used as a filtering step before using phylogenetic methods and provides a way to interpret BLAST results using more information than provided by E-values and bit-scores alone.

     

Molecular phylogenetics of Meliaceae (Sapindales) based on nuclear and plastid DNA sequences

Phylogenetic analyses of Meliaceae, including representatives of all four currently recognized subfamilies and all but two tribes (32 genera and 35 species, respectively), were carried out using DNA sequence data from three regions: plastid genes rbcL, matK (partial), and nuclear 26S rDNA (partial). Individual and combined phylogenetic analyses were performed for the rbcL, matK, and 26S rDNA data sets. Although the percentage of informative characters is highest in the segment of matK sequenced, rbcL provides the greatest number of informative characters of the three regions, resulting in the best resolved trees. Results of parsimony analyses support the recognition of only two subfamilies (Melioideae and Swietenioideae), which are sister groups. Melieae are the only tribe recognized previously that are strongly supported as monophyletic. The members of the two small monogeneric subfamilies, Quivisianthe and Capuronianthus, fall within Melioideae and Swietenioideae, respectively, supporting their taxonomic inclusion in these groups. Furthermore, the data indicate a close relationship between Aglaieae and Guareeae and a possible monophyletic origin of Cedreleae of Swietenioideae. For Trichilieae (Melioideae) and Swietenieae (Swietenioideae) lack of monophyly is indicated.     

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.     

Depth-related patterns of meiofauna on the Indian continental shelf are conserved at reduced taxonomic resolution

Taxonomic sufficiency (TS) has been used in impact assessment studies of various pollution effects on marine benthic communities and found appropriate to identify the effects of pollution on marine communities. Cost, in terms of the expertise and time needed to identify organisms, increases with the level of taxonomic accuracy. Recently, TS has been adopted to study spatial patterns of macrobenthic community structure. In order to accept TS as a routine approach in wider benthic studies, it needs to be proved valid for various taxa and in geographically different areas. The present study investigates the value of TS in meiofaunal nematodes by analyzing an extensive data set based on samples collected from a wide geographical area covering a large depth gradient. For this study, samples were collected from every degree square of the western Indian continental shelf (7°–22°N latitudes). Our high resolution data showed that with increase in depth, nematode species richness and diversity decreased and communities showed significant variation between shallow and deeper waters. The present study tests whether lower taxonomic resolution nematode data can explain community shifts along a depth gradient in a similar way to species level data from the same data set. Meiofauna have often been neglected from benthic studies, and most attention has been given to macrofauna. This is mainly due to the difficulty in the taxonomic identification of meiofauna. The results of this study based on univariate and multivariate analyses support the use of family level data of nematodes to explain some aspects of depth variation in a similar way to species level data.     

Corroboration among Data Sets in Simultaneous Analysis: Hidden Support for Phylogenetic Relationships among Higher Level Artiodactyl Taxa

In the taxonomic congruence approach to systematics, data sets are analyzed separately, and corroboration among data sets is indicated by replicated components in topologies derived from the separate analyses. By contrast, in the total evidence and conditional combination approaches, characters from different data sets are mixed in combined phylogenetic analyses. In optimal topologies derived from such simultaneous analyses, support for a particular node may be attributed to one, some, or all of the individual data sets. Partitioned branch support (PBS) is one technique for describing the distribution of character support and conflict among data sets in simultaneous analysis. PBS is analogous to branch support (BS), but recognizes hidden support and conflicts that emerge with the combination of characters from different data sets. For both BS and PBS, support for a particular node is interpreted as the difference in cost between optimal and suboptimal topologies. A different measure, the clade stability index (CSI), assesses the robustness of a particular node through the successive removal of characters. Here, we introduce variations of the CSI, the data set removal index (DRI) and nodal data set influence (NDI), that indicate the stability of a particular node to the removal of entire data sets. Like PBS, the DRI and NDI summarize the influence of different data sets in simultaneous analysis. However, because these new methods and PBS use different perturbations to assess stability, DRI and NDI scores do not always predict PBS scores and vice versa. In this report, the DRI and NDI are compared to PBS and taxonomic congruence in a cladistic analysis of 17 data sets for Artiodactyla (Mammalia). Five indices of hidden support and conflict are defined and applied to the combined artiodactyl character set. These measures identify substantial hidden support for controversial relationships within Artiodactyla. Hidden character support is ignored in the taxonomic congruence approach to systematics, but the DRI, NDI, and PBS utilize this cryptic information in estimates of support among data sets for a given node.     

Genetic diversity and taxonomy: a reassessment of species designation in tuatara (<Emphasis Type="Italic">Sphenodon</Emphasis>: Reptilia)

The identification of species boundaries for allopatric populations is important for setting conservation priorities and can affect conservation management decisions. Tuatara (Sphenodon) are the only living members of the reptile order Sphenodontia and are restricted to islands around New Zealand that are free of introduced mammals. We present new data of microsatellite DNA diversity and substantially increased mtDNA sequence for all 26 sampled tuatara populations. We also re-evaluate existing allozyme data for those populations, and together use them to examine the taxonomic status of those populations. Although one could interpret the data to indicate different taxonomic designations, we conclude that, contrary to current taxonomy, Sphenodon is best described as a single species that contains distinctive and important geographic variants. We also examine amounts of genetic variation within populations and discuss the implications of these findings for the conservation management of this iconic taxon.     

Molecular Barcoding of Aquatic Oligochaetes: Implications for Biomonitoring

Aquatic oligochaetes are well recognized bioindicators of quality of sediments and water in watercourses and lakes. However, the difficult taxonomic determination based on morphological features compromises their more common use in eco-diagnostic analyses. To overcome this limitation, we investigated molecular barcodes as identification tool for broad range of taxa of aquatic oligochaetes. We report 185 COI and 52 ITS2 rDNA sequences for specimens collected in Switzerland and belonging to the families Naididae, Lumbriculidae, Enchytraeidae and Lumbricidae. Phylogenetic analyses allowed distinguishing 41 lineages separated by more than 10 % divergence in COI sequences. The lineage distinction was confirmed by Automatic Barcode Gap Discovery (ABGD) method and by ITS2 data. Our results showed that morphological identification underestimates the oligochaete diversity. Only 26 of the lineages could be assigned to morphospecies, of which seven were sequenced for the first time. Several cryptic species were detected within common morphospecies. Many juvenile specimens that could not be assigned morphologically have found their home after genetic analysis. Our study showed that COI barcodes performed very well as species identifiers in aquatic oligochaetes. Their easy amplification and good taxonomic resolution might help promoting aquatic oligochaetes as bioindicators for next generation environmental DNA biomonitoring of aquatic ecosystems.     

Novel DNA markers reveal for taxonomic characterization and identification of the Fusarium fungi species

RAPD analysis for ten F. sporotrichioides strains of different geographical origin was done for DNA loci, potentially suitable as a new markers for taxonomic characterization and identification of toxigenic Fusarium fungi. Three selected monomorphic fragments--products of amplification with one of standard RAPD primers were sequenced that allowed creating SCAR markers for identification of Fusarium fungi on the species group level with similar profiles of produced mycotoxins.     

Significance of hind wing morphology in distinguishing genera and species of cantharid beetles with a geometric morphometric analysis

There remain some difficulties in delimitation of related genera or sibling species for cantharid beetles, because the traditionally taxonomic method and morphological characters have not been updated or introduced. In the present study, we firstly use the landmark-based geometric morphometrics to analyze and compare the hind wings of nine species belonging to three genera of Cantharinae to ascertain whether this approach may be used as a reliable method in the study of the taxonomy of this group. The results show that the shape differences of the hind wings among genera seem more variable than that within each genus, and the variations for each species are different from one another, as shown in the principal component analyses. And the canonical variates analyses show that there are significant differences among the genera and the species of each genus, which demonstrates that the hind wing shape can be diagnostic for both generic and specific identification of the cantharid beetles. This study sheds new light into clarifying the taxonomic uncertainties of Cantharidae, and lays a foundation for further studies on the evolution of the cantharid hind wing shape.     

An integrative approach to species discovery in odonates: from character‐based DNA barcoding to ecology

Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision‐making. Such an approach can enhance both species identification and species discovery. The character‐based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA‐barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.     

Characterization of Lake Michigan coastal lakes using zooplankton assemblages

Zooplankton assemblages and water quality were examined bi-weekly from 17 April to 19 October 1998 in 11 northeastern Lake Michigan coastal lakes of similar origin but varied in trophic status and limnological condition. All lakes were within or adjacent to Sleeping Bear Dunes National Lakeshore, Michigan. Zooplankton (principally microcrustaceans and rotifers) from triplicate Wisconsin net (80 μm) vertical tows taken at each lake's deepest location were analyzed. Oxygen–temperature–pH–specific conductivity profiles and surface water quality were concurrently measured. Bray-Curtis similarity analysis showed small variations among sample replicates but large temporal differences. The potential use of zooplankton communities for environmental lake comparisons was evaluated by means of BIOENV (Primer 5.1) and principal component analyses. Zooplankton analyzed at the lowest identified taxonomic level yielded greatest sensitivity to limnological variation. Taxonomic and ecological aggregations of zooplankton data performed comparably, but less well than the finest taxonomic analysis. Secchi depth, chlorophyll a, and sulfate concentrations combined to give the best correlation with patterns of variation in the zooplankton data set. Principal component analysis of these variables revealed trophic status as the most influential major limnological gradient among the study lakes. Overall, zooplankton abundance was an excellent indicator of variation in trophic status.     

The origins of an important cactus crop, Opuntia ficus-indica (Cactaceae): new molecular evidence

Opuntia ficus-indica is a long-domesticated cactus crop that is important in agricultural economies throughout arid and semiarid parts of the world. The biogeographic and evolutionary origins of this species have been obscured through ancient and widespread cultivation and naturalization. The origin of O. ficus-indica is investigated through the use of Bayesian phylogenetic analyses of nrITS DNA sequences. These analyses support the following hypotheses: that O. ficus-indica is a close relative of a group of arborescent, fleshy-fruited prickly pears from central and southern Mexico; that the center of domestication for this species is in central Mexico; and that the taxonomic concept of O. ficus-indica may include clones derived from multiple lineages and therefore be polyphyletic.     

交互式检索表提升分类学研究与应用效率:以兰科植物为例

与传统的连续检索表相比,基于计算机等数字终端的多通道交互式检索表具有很多优势,如易于携带、实时更新、界面直观、使用方便、用户广泛、产生新数据集且便于研究以及能够推动分类学知识的普及等。本文以兰科植物为例介绍了交互式检索表的特点、程序建立和应用。兰科植物是生物多样性保育的旗舰类群,具有很强的观赏和药用价值,但中国兰科植物种类繁多,分类困难。我们在自主研发交互式检索表软件MagIden的基础上,以《中国植物志》和Flora of China的描述为依据,制作了中国兰科植物交互式电子检索表,为分类学研究者和爱好者提供了一个新的、鉴定中国兰科植物的中英文双语工具;同时,交互式检索表生成的MVSP和Bayes形态特征数据集可用于聚类分析和分支分类学研究。交互式检索表在数据积累、更新和分析利用等方面具有独特的优势,是提高传统植物分类学研究与应用效率的重要工具。