One species in eight: DNA barcodes from type specimens resolve a taxonomic quagmire

Each holotype specimen provides the only objective link to a particular Linnean binomen. Sequence information from them is increasingly valuable due to the growing usage of DNA barcodes in taxonomy. As type specimens are often old, it may only be possible to recover fragmentary sequence information from them. We tested the efficacy of short sequences from type specimens in the resolution of a challenging taxonomic puzzle: the Elachista dispunctella complex which includes 64 described species with minuscule morphological differences. We applied a multistep procedure to resolve the taxonomy of this species complex. First, we sequenced a large number of newly collected specimens and as many holotypes as possible. Second, we used all >400 bp examine species boundaries. We employed three unsupervised methods (BIN, ABGD, GMYC) with specified criteria on how to handle discordant results and examined diagnostic bases from each delineated putative species (operational taxonomic units, OTUs). Third, we evaluated the morphological characters of each OTU. Finally, we associated short barcodes from types with the delineated OTUs. In this step, we employed various supervised methods, including distance‐based, tree‐based and character‐based. We recovered 658 bp barcode sequences from 194 of 215 fresh specimens and recovered an average of 141 bp from 33 of 42 holotypes. We observed strong congruence among all methods and good correspondence with morphology. We demonstrate potential pitfalls with tree‐, distance‐ and character‐based approaches when associating sequences of varied length. Our results suggest that sequences as short as 56 bp can often provide valuable taxonomic information. The results support significant taxonomic oversplitting of species in the Elachista dispunctella complex.     

Delineating Species with DNA Barcodes: A Case of Taxon Dependent Method Performance in Moths

The accelerating loss of biodiversity has created a need for more effective ways to discover species. Novel algorithmic approaches for analyzing sequence data combined with rapidly expanding DNA barcode libraries provide a potential solution. While several analytical methods are available for the delineation of operational taxonomic units (OTUs), few studies have compared their performance. This study compares the performance of one morphology-based and four DNA-based (BIN, parsimony networks, ABGD, GMYC) methods on two groups of gelechioid moths. It examines 92 species of Finnish Gelechiinae and 103 species of Australian Elachistinae which were delineated by traditional taxonomy. The results reveal a striking difference in performance between the two taxa with all four DNA-based methods. OTU counts in the Elachistinae showed a wider range and a relatively low (ca. 65%) OTU match with reference species while OTU counts were more congruent and performance was higher (ca. 90%) in the Gelechiinae. Performance rose when only monophyletic species were compared, but the taxon-dependence remained. None of the DNA-based methods produced a correct match with non-monophyletic species, but singletons were handled well. A simulated test of morphospecies-grouping performed very poorly in revealing taxon diversity in these small, dull-colored moths. Despite the strong performance of analyses based on DNA barcodes, species delineated using single-locus mtDNA data are best viewed as OTUs that require validation by subsequent integrative taxonomic work.     

Metabarcoding of freshwater invertebrates to detect the effects of a pesticide spill

Biomonitoring underpins the environmental assessment of freshwater ecosystems and guides management and conservation. Current methodology for surveys of (macro)invertebrates uses coarse taxonomic identification where species‐level resolution is difficult to obtain. Next‐generation sequencing of entire assemblages (metabarcoding) provides a new approach for species detection, but requires further validation. We used metabarcoding of invertebrate assemblages with two fragments of the cox1 “barcode” and partial nuclear ribosomal (SSU) genes, to assess the effects of a pesticide spill in the River Kennet (southern England). Operational taxonomic unit (OTU) recovery was tested under 72 parameters (read denoising, filtering, pair merging and clustering). Similar taxonomic profiles were obtained under a broad range of parameters. The SSU marker recovered Platyhelminthes and Nematoda, missed by cox1, while Rotifera were only amplified with cox1. A reference set was created from all available barcode entries for Arthropoda in the BOLD database and clustered into OTUs. The River Kennet metabarcoding produced matches to 207 of these reference OTUs, five times the number of species recognized with morphological monitoring. The increase was due to the following: greater taxonomic resolution (e.g., splitting a single morphotaxon “Chironomidae” into 55 named OTUs); splitting of Linnaean binomials into multiple molecular OTUs; and the use of a filtration‐flotation protocol for extraction of minute specimens (meiofauna). Community analyses revealed strong differences between “impacted” vs. “control” samples, detectable with each gene marker, for each major taxonomic group, and for meio‐ and macrofaunal samples separately. Thus, highly resolved taxonomic data can be extracted at a fraction of the time and cost of traditional nonmolecular methods, opening new avenues for freshwater invertebrate biodiversity monitoring and molecular ecology.     

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo‐West Pacific uncovered by DNA barcoding

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty‐five operational taxonomic units (OTUs) were observed across the Indo‐West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.     

Algorithmic single‐locus species delimitation: effects of sampling effort,variation and nonmonophyly in four methods and 1870 species of beetles

The vast number of undescribed species and the fast rate of biodiversity loss call for new approaches to speed up alpha taxonomy. A plethora of methods for delimiting species or operational taxonomic units (OTUs) based on sequence data have been published in recent years. We test the ability of four delimitation methods (BIN, ABGD, GMYC, PTP) to reproduce established species boundaries on a carefully curated DNA barcode data set of 1870 North European beetle species. We also explore how sampling effort, intraspecific variation, nearest neighbour divergence and nonmonophyly affect the OTU delimitations. All methods produced approximately 90% identity between species and OTUs. The effects of variation and sampling differed between methods. ABGD was sensitive to singleton sequences, while GMYC showed tendencies for oversplitting. The best fit between species and OTUs was achieved using simple rules to find consensus between discordant OTU delimitations. Using several approaches simultaneously allows the methods to compensate for each other's weaknesses. Barcode‐based OTU‐picking is an efficient way to delimit putative species from large data sets where the use of more sophisticated methods based on multilocus or genomic data is not feasible.     

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.     

Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods

Metabarcoding has the potential to become a rapid, sensitive, and effective approach for identifying species in complex environmental samples. Accurate molecular identification of species depends on the ability to generate operational taxonomic units (OTUs) that correspond to biological species. Due to the sometimes enormous estimates of biodiversity using this method, there is a great need to test the efficacy of data analysis methods used to derive OTUs. Here, we evaluate the performance of various methods for clustering length variable 18S amplicons from complex samples into OTUs using a mock community and a natural community of zooplankton species. We compare analytic procedures consisting of a combination of (1) stringent and relaxed data filtering, (2) singleton sequences included and removed, (3) three commonly used clustering algorithms (mothur, UCLUST, and UPARSE), and (4) three methods of treating alignment gaps when calculating sequence divergence. Depending on the combination of methods used, the number of OTUs varied by nearly two orders of magnitude for the mock community (60–5068 OTUs) and three orders of magnitude for the natural community (22–22191 OTUs). The use of relaxed filtering and the inclusion of singletons greatly inflated OTU numbers without increasing the ability to recover species. Our results also suggest that the method used to treat gaps when calculating sequence divergence can have a great impact on the number of OTUs. Our findings are particularly relevant to studies that cover taxonomically diverse species and employ markers such as rRNA genes in which length variation is extensive.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

Application of principal components analyses with constant character number in a study of the Bulbostylis/Fimbristylis (Cyperaceae) complex in Nigeria

Interrelationships between OTUs (operational taxonomic units) were assessed through comparisons based on a constant character number, using principal components analysis. This approach results in a uniform treatment of OTUs, despite variations in the number of definite entries for character states that could validly be made. In addition, the risk of distortion of the ordination scatter as a result of a bimodal frequency distribution of the number of characters available for comparisons is minimized. Appropriately applied, the approach also provides indications of the consistency of the features of the ordinations and reveals where more caution in interpretation is advisable.
The main analysis divided the OTUs into two unequal-sized groups, with a few intermediates; these were concluded to be more closely allied to the large than the small group. The significance of these groupings, in terms of their species contents, is discussed in relation to the method and to recent taxonomic treatments of the Bulbostylis/Fimbristylis complex. Some taxonomic conclusions must be tentative because of the limitations of the sample.     

Documenting neotropical diversity of phoronids with DNA barcoding of planktonic larvae

Phoronid larvae, actinotrochs, are beautiful and complicated organisms which have attracted as much, if not more, attention than their adult forms. We collected actinotrochs from the waters of the Pacific and Caribbean coasts of Panama, and used DNA barcoding of mtCOI, as well as 16S and 18S sequences, to estimate the diversity of phoronids in the region. We discovered three operational taxonomic units (OTUs) in the Bay of Panama on the Pacific coast and four OTUs in Bocas del Toro on the Caribbean coast. Not only did all OTUs differ from each other by >10% pairwise distance in COI, but they also differed from all phoronid sequences in GenBank, including the four species for which adults have been reported for the Pacific of Panama, Phoronopsis harmeri, Phoronis psammophila, Phoronis muelleri, and Phoronis hippocrepia. In each ocean region, one common OTU was more abundant and occurred more frequently than other OTUs in our samples. The other five OTUs were relatively rare, with only one to three individuals collected during the entire project. Species accumulation curves were relatively flat but suggest that at least one more species is likely to be present at each site. Actinotrochs from the seven sequenced OTUs had morphologies typical of species with non‐brooded planktotrophic development and, in some cases, may be distinguished by differences in pigmentation and the arrangement of blood masses. We found one larva with morphology typical of brooded planktotrophic larvae for which sequencing failed, bringing the total number of potential species detected to eight and representing >50% of the adult species currently recognized globally.     

Fungal communities on decaying leaves in streams: a comparison of two leaf species

Decomposition of leaf litter is a microbial mediated process that helps to transfer energy and nutrients from leaves to higher trophic levels in woodland streams. Generally, aquatic hyphomycetes are viewed as the major fungal group responsible for leaf litter decomposition. In this study, traditional microscopic examination (based on identification of released conidia) and phylogenetic analysis of 18S rRNA genes from cultivated fungi were used to compare fungal community composition on decomposing leaves of two species (sugar maple and white oak) from a NE Ohio stream. No significant differences were found in sporulation rates between maple and oak leaves and both had similar species diversity. From the 18S rRNA gene sequence data, identification was achieved for 12 isolates and taxonomic affiliation of 12 of the remaining 14 isolates could be obtained. A neighbor-joining tree (with bootstrap values) was constructed to examine the taxonomic distribution of the isolates relative to sequences of known operational taxonomic units (OTUs). Surprisingly, only 2 of the isolates obtained were aquatic hyphomycetes based on phylogenetic analysis. Overall, there were no differences between the two leaf types and a higher diversity was observed via culturing and subsequent 18S rRNA gene sequencing than by conidia staining. These differences resulted from the fact that traditional microscopy provides estimates of aquatic hyphomycete diversity while the other approach revealed the presence of both aquatic hyphomycete and non-aquatic hyphomycete taxa. The presence of this broad array of taxa suggests that the role of aquatic hyphomycetes relative to other fungi be re-evaluated. Even though the functional role of these non-aquatic hyphomycetes taxa is unknown, their presence and diversity demonstrates the need to delve further into fungal community structure on decomposing leaves.     

Hiding deep in the trees: discovery of divergent mitochondrial lineages in Malagasy chameleons of the Calumma nasutum group

WE CONDUCTED A COMPREHENSIVE MOLECULAR PHYLOGENETIC STUDY FOR A GROUP OF CHAMELEONS FROM MADAGASCAR (CHAMAELEONIDAE: Calumma nasutum group, comprising seven nominal species) to examine the genetic and species diversity in this widespread genus. Based on DNA sequences of the mitochondrial gene (ND2) from 215 specimens, we reconstructed the phylogeny using a Bayesian approach. Our results show deep divergences among several unnamed mitochondrial lineages that are difficult to identify morphologically. We evaluated lineage diversification using a number of statistical phylogenetic methods (general mixed Yule-coalescent model; SpeciesIdentifier; net p-distances) to objectively delimit lineages that we here consider as operational taxonomic units (OTUs), and for which the taxonomic status remains largely unknown. In addition, we compared molecular and morphological differentiation in detail for one particularly diverse clade (the C. boettgeri complex) from northern Madagascar. To assess the species boundaries within this group we used an integrative taxonomic approach, combining evidence from two independent molecular markers (ND2 and CMOS), together with genital and other external morphological characters, and conclude that some of the newly discovered OTUs are separate species (confirmed candidate species, CCS), while others should best be considered as deep conspecific lineages (DCLs). Our analysis supports a total of 33 OTUs, of which seven correspond to described species, suggesting that the taxonomy of the C. nasutum group is in need of revision.     

3种杓兰属植物菌根真菌系统发育和多样性分析

兰科植物菌根真菌(Orchid mycorrhizal fungi, OrMF)在兰科植物种子萌发和后续生长发育过程中具有重要作用。该研究采用培养(菌丝团分离)和非培养(克隆文库)2种方法获得同一栖息地3种不同杓兰属植物根中菌根真菌ITS序列并划分可操作分类单元(Operational taxonomic units, OTUs),分析其系统发育关系和多样性。结果表明:(1)所有根段中都有菌丝团定植,共分离出菌根真菌64株,其中63株为胶膜菌科(Tulasnellaceae)真菌,1株为角担菌科(Ceratobasidiaceae)真菌;可划分为7个OUT,每个OTU代表菌株的菌丝都能形成OrMF典型的近球形或椭球形链状排列的念珠状细胞;分离出来的菌根真菌均为无性型菌丝且不产生无性孢子。(2) 非培养法得到的3种杓兰属植物的根中OrMF分别隶属于胶膜菌科(Tulasnellaceae),腊壳菌科(Sebacinaceae)、角担菌科(Ceratobasidiaceae)和革菌科(Thelephoraceae),其中胶膜菌科OTU在种类和数量上占有绝对优势,培养和非培养2种方法得到的OrMF OTU类型和数量均为西藏杓兰(Cypripedium tibeticum)>无苞杓兰(C. flavum)>黄花杓兰(C. bardolphianum),但培养法少于非培养法。(3)对胶膜菌进行系统发育分析显示,优势和非优势OTU均分布在系统发育树的3个不同分支上,这种与多种亲缘关系较远的OrMF共生的现象可能与杓兰属植物对环境的适应性有关,且不同杓兰的OrMF物种丰富度没有显著差异,但群落结构存在差异。     

Identifying airborne fungi in Seoul,Korea using metagenomics

Fungal spores are widespread and common in the atmosphere. In this study, we use a metagenomic approach to study the fungal diversity in six total air samples collected from April to May 2012 in Seoul, Korea. This springtime period is important in Korea because of the peak in fungal spore concentration and Asian dust storms, although the year of this study (2012) was unique in that were no major Asian dust events. Clustering sequences for operational taxonomic unit (OTU) identification recovered 1,266 unique OTUs in the combined dataset, with between 223?96 OTUs present in individual samples. OTUs from three fungal phyla were identified. For Ascomycota, Davidiella (anamorph: Cladosporium) was the most common genus in all samples, often accounting for more than 50% of all sequences in a sample. Other common Ascomycota genera identified were Alternaria, Didymella, Khuskia, Geosmitha, Penicillium, and Aspergillus. While several Basidiomycota genera were observed, Chytridiomycota OTUs were only present in one sample. Consistency was observed within sampling days, but there was a large shift in species composition from Ascomycota dominant to Basidiomycota dominant in the middle of the sampling period. This marked change may have been caused by meteorological events. A potential set of 40 allergyinducing genera were identified, accounting for a large proportion of the diversity present (22.5?7.2%). Our study identifies high fungal diversity and potentially high levels of fungal allergens in springtime air of Korea, and provides a good baseline for future comparisons with Asian dust storms.     

Comparison of morphological,DNA barcoding,and metabarcoding characterizations of freshwater nematode communities

Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single‐specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification.     

DNA barcodes as a tool in biodiversity research: testing pre-existing taxonomic hypotheses in Delphic Apollo butterflies (Lepidoptera,Papilionidae)

Numerous studies have demonstrated that DNA barcoding is an effective tool for detecting DNA clusters, which can be viewed as operational taxonomic units (OTUs), useful for biodiversity research. Frequently, the OTUs in these studies remained unnamed, not connected with pre-existing taxonomic hypotheses, and thus did not really contribute to feasible estimation of species number and adjustment of species boundaries. For the majority of organisms, taxonomy is very complicated with numerous, often contradictory interpretations of the same characters, which may result in several competing checklists using different specific and subspecific names to describe the same sets of populations. The highly species-rich genus Parnassius (Lepidoptera: Papilionidae) is but one example, such as several mutually exclusive taxonomic systems have been suggested to describe the phenotypic diversity found among its populations. Here we provide an explicit flow chart describing how the DNA barcodes can be combined with the existing knowledge of morphology-based taxonomy and geography (sympatry versus allopatry) of the studied populations in order to support, reject or modify the pre-existing taxonomic hypotheses. We then apply this flow chart to reorganize the taxa within the Parnassius delphius species group, solving long-standing taxonomic problems.     

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.     

Untangling taxonomy: a DNA barcode reference library for Canadian spiders

Approximately 1460 species of spiders have been reported from Canada, 3% of the global fauna. This study provides a DNA barcode reference library for 1018 of these species based upon the analysis of more than 30 000 specimens. The sequence results show a clear barcode gap in most cases with a mean intraspecific divergence of 0.78% vs. a minimum nearest‐neighbour (NN) distance averaging 7.85%. The sequences were assigned to 1359 Barcode index numbers (BINs) with 1344 of these BINs composed of specimens belonging to a single currently recognized species. There was a perfect correspondence between BIN membership and a known species in 795 cases, while another 197 species were assigned to two or more BINs (556 in total). A few other species (26) were involved in BIN merges or in a combination of merges and splits. There was only a weak relationship between the number of specimens analysed for a species and its BIN count. However, three species were clear outliers with their specimens being placed in 11–22 BINs. Although all BIN splits need further study to clarify the taxonomic status of the entities involved, DNA barcodes discriminated 98% of the 1018 species. The present survey conservatively revealed 16 species new to science, 52 species new to Canada and major range extensions for 426 species. However, if most BIN splits detected in this study reflect cryptic taxa, the true species count for Canadian spiders could be 30–50% higher than currently recognized.     

NUMERICAL TAXONOMY OF LIMNANTHACEAE

A numerical taxonomic study of the angiosperm family Limnanthaceae is presented. The 21 OTUs include all taxa recognized in a recent monograph of Limnanthes (Mason, 1952), plus Floerkea proserpinacoides, two undescribed Limnanthes species, and two artificial interspecific F₁ Limnanthes hybrids. One species (L. montana) was treated as two OTUs: measurements for one OTU were taken from field collections and for the other, from garden-grown plants. A maximum of 35 characters was used in the numerical analyses. Raw data for each character were given equal weighting by condensation. Coefficients of resemblance were established using the mean character difference (MCD) method. Tryon's clustering technique (called key communality cluster analysis) was used. Three numerical analyses of Limnanthaceae were made: one with all 35 characters (T analysis), one with 18 floral characters (F analysis), and one with 17 pre- or post-anthesis or vegetative characters (V analysis). Relative positions of OTUs in the T and F analyses are substantially different from those in a conventional taxonomic treatment of the family. The V analysis shows many differences from the T and F analyses. In all analyses the two OTUs representing L. montana were closer to other OTUs than to each other. The two hybrid OTUs showed greater resemblance to one parent than to the other and in two analyses one hybrid was not intermediate between the parents. Similarities between the T and F analyses are explained on the basis of the predominance of floral characters in both. The differences between these two analyses on the one hand and the V analysis and phylogenetic arrangement of the family on the other are attributed to parallelisms in the evolution of floral characters associated with iucreasing autogamy in each of the two phyletic lines in the family. Analyses emphasizing floral characters will place evolutionarily remotely related outcrossing OTUs together and unrelated, autogamous OTUs together. Using characters that are mostly unassociated with the breeding system the positions of OTUs in a numerical analysis are similar to their positions in a phylogenetic arrangement. The V analysis agrees best with suggestions concerning phylogenetic affinities among the taxa in the family, and provides a useful context for the generation of further evolutionary hypotheses.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.