MOTU analysis of ichthyoplankton biodiversity in the upper Yangtze River,China

Identification of ichthyoplankton is difficult because fish during early life stages often lack stable morphological characteristics; such difficulty in species identification can be a major hindrance in conducting ichthyoplankton surveys for fish biodiversity investigations. Here, we evaluated the feasibility of a molecular operational taxonomic unit (MOTU) approach for ichthyoplankton investigations, and describe fish biodiversity in the Jinshajiang section of the upper Yangtze River, China. The MOTUs were established by grouping specimens diverging less than 1.00% Kimura two‐parameter (K2P) distance units from their nearest neighbor within the same MOTU, based on previous work on between‐species divergences of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Taxonomic assignment of the MOTUs was performed by comparing the MOTU sequences with the COI sequences of taxonomic species. Sixty‐eight MOTUs were inferred from 818 COI sequences of ichthyoplankton in the Jinshajiang river section. Among those, one MOTU was composed of two identified taxonomic species, and each of the other MOTUs was linked to a single, identified taxonomic species. Only 26 MOTUs were successfully identified to taxonomic species due to the limited reference database. Our results demonstrate that the MOTU approach can be applied successfully for analyzing biodiversity and identifying species of freshwater ichthyoplankton. Compared with previous ichthyoplankton investigations the richness of ichthyoplankton was very high. High diversity of ichthyoplankton noted in our study suggests that the Jinshajiang section should be an important target for fish biodiversity conservation in the Yangtze River.     

Optimal sequence similarity thresholds for clustering of molecular operational taxonomic units in DNA metabarcoding studies

Clustering approaches are pivotal to handle the many sequence variants obtained in DNA metabarcoding data sets, and therefore they have become a key step of metabarcoding analysis pipelines. Clustering often relies on a sequence similarity threshold to gather sequences into molecular operational taxonomic units (MOTUs), each of which ideally represents a homogeneous taxonomic entity (e.g., a species or a genus). However, the choice of the clustering threshold is rarely justified, and its impact on MOTU over-splitting or over-merging even less tested. Here, we evaluated clustering threshold values for several metabarcoding markers under different criteria: limitation of MOTU over-merging, limitation of MOTU over-splitting, and trade-off between over-merging and over-splitting. We extracted sequences from a public database for nine markers, ranging from generalist markers targeting Bacteria or Eukaryota, to more specific markers targeting a class or a subclass (e.g., Insecta, Oligochaeta). Based on the distributions of pairwise sequence similarities within species and within genera, and on the rates of over-splitting and over-merging across different clustering thresholds, we were able to propose threshold values minimizing the risk of over-splitting, that of over-merging, or offering a trade-off between the two risks. For generalist markers, high similarity thresholds (0.96–0.99) are generally appropriate, while more specific markers require lower values (0.85–0.96). These results do not support the use of a fixed clustering threshold. Instead, we advocate careful examination of the most appropriate threshold based on the research objectives, the potential costs of over-splitting and over-merging, and the features of the studied markers.     

Environmental and spatial characterisation of an unknown fauna using DNA sequencing – an example with Himalayan Hydropsychidae (Insecta: Trichoptera)

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MOTUs, Morphology, and Biodiversity Estimation: A Case Study Using Nematodes of the Suborder Criconematina and a Conserved 18S DNA Barcode

DNA barcodes are increasingly used to provide an estimate of biodiversity for small, cryptic organisms like nematodes. Nucleotide sequences generated by the barcoding process are often grouped, based on similarity, into molecular operational taxonomic units (MOTUs). In order to get a better understanding of the taxonomic resolution of a 3' 592-bp 18S rDNA barcode, we have analyzed 100 MOTUs generated from 214 specimens in the nematode suborder Criconematina. Previous research has demonstrated that the primer set for this barcode reliably amplifies all nematodes in the Phylum Nematoda. Included among the Criconematina specimens were 25 morphologically described species representing 12 genera. Using the most stringent definition of MOTU membership, where a single nucleotide difference is sufficient for the creation of a new MOTU, it was found that an MOTU can represent a subgroup of a species (e.g. Discocriconemella limitanea), a single species (Bakernema inaequale), or a species complex (MOTU 76). A maximum likelihood phylogenetic analysis of the MOTU dataset generated four major clades that were further analyzed by character-based barcode analysis. Fourteen of the 25 morphologically identified species had at least one putative diagnostic nucleotide identified by this character-based approach. These diagnostic nucleotides could be useful in biodiversity assessments when ambiguous results are encountered in database searches that use a distance-based metric for nucleotide sequence comparisons. Information and images regarding specimens examined during this study are available online.     

High-Throughput Sequencing—The Key to Rapid Biodiversity Assessment of Marine Metazoa?

The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of the V1-V2 region was used to analyze two pooled samples of DNA, one comprising 118 and the other 37 morphologically identified species, and one natural sample taken directly from a North Sea zooplankton community. A DNA reference library comprising all species represented in the pooled samples was created by Sanger sequencing, and this was then used to determine the optimal similarity threshold for species delineation. The optimal threshold was found at 99% species similarity, with 85% identification success. Pyrosequencing was able to identify between fewer species: 67% and 78% of the species in the two pooled samples. Also, a large number of sequences for three species that were not included in the pooled samples were amplified by pyrosequencing, suggesting preferential amplification of some genotypes and the sensitivity of this approach to even low levels of contamination. Conversely, metagenetic analysis of the natural zooplankton sample identified many more species (particularly gelatinous zooplankton and meroplankton) than morphological analysis of a formalin-fixed sample from the same sampling site, suggesting an increased level of taxonomic resolution with pyrosequencing. The study demonstrated that, based on the V1-V2 region, 454 sequencing does not provide accurate species differentiation and reliable taxonomic classification, as it is required in most biodiversity monitoring. The analysis of artificially prepared samples indicated that species detection in pyrosequencing datasets is complicated by potential PCR-based biases and that the V1-V2 marker is poorly resolved for some taxa.     

Investigating Microbial Eukaryotic Diversity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of 18S rRNA Genes

Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.     

Fast Census of Moth Diversity in the Neotropics: A Comparison of Field-Assigned Morphospecies and DNA Barcoding in Tiger Moths

The morphological species delimitations (i.e. morphospecies) have long been the best way to avoid the taxonomic impediment and compare insect taxa biodiversity in highly diverse tropical and subtropical regions. The development of DNA barcoding, however, has shown great potential to replace (or at least complement) the morphospecies approach, with the advantage of relying on automated methods implemented in computer programs or even online rather than in often subjective morphological features. We sampled moths extensively for two years using light traps in a patch of the highly endangered Atlantic Forest of Brazil to produce a nearly complete census of arctiines (Noctuoidea: Erebidae), whose species richness was compared using different morphological and molecular approaches (DNA barcoding). A total of 1,075 barcode sequences of 286 morphospecies were analyzed. Based on the clustering method Barcode Index Number (BIN) we found a taxonomic bias of approximately 30% in our initial morphological assessment. However, a morphological reassessment revealed that the correspondence between morphospecies and molecular operational taxonomic units (MOTUs) can be up to 94% if differences in genitalia morphology are evaluated in individuals of different MOTUs originated from the same morphospecies (putative cases of cryptic species), and by recording if individuals of different genders in different morphospecies merge together in the same MOTU (putative cases of sexual dimorphism). The results of two other clustering methods (i.e. Automatic Barcode Gap Discovery and 2% threshold) were very similar to those of the BIN approach. Using empirical data we have shown that DNA barcoding performed substantially better than the morphospecies approach, based on superficial morphology, to delimit species of a highly diverse moth taxon, and thus should be used in species inventories.     

Next Generation Sequencing Reveals the Hidden Diversity of Zooplankton Assemblages

Background

Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel.

Methodology/Principle Findings

Plankton net hauls (200 µm) were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups.

Conclusions

Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may become increasingly attractive in future if sequence reference libraries of accurately identified individuals are better populated.     

Clustering of fungal community internal transcribed spacer sequence data obscures taxonomic diversity

Next‐generation DNA sequencing has enabled a rapid expansion in the size of molecular fungal ecology studies employing the nuclear internal transcribed spacer (ITS) region. Many sequence‐processing pipelines and protocols require sequence clustering to generate operational taxonomic units (OTUs) based on sequence similarity as a step to reduce total data quantity and complexity prior to taxonomic assignment. However, the consequences of ITS sequence clustering in regard to sample taxonomic coverage have not been carefully examined. Here we demonstrate that typically used clustering thresholds for fungal ITS sequences result in statistically significant losses in taxonomic coverage. Analyses using environmentally derived fungal sequences indicated an average of 3.1% of species went undetected (P < 0.05) if the sequences were denoised and clustered at a 97% threshold prior to taxonomic assignment. Additionally, an in silico analysis using a reference fungal ITS database suggested that approximately 25% of species went undetected if the sequences were clustered prior to taxonomic assignment. Finally, analysis of sequences derived from pure‐cultured fungal isolates of known identity indicated sequence denoising and clustering were not critical in improving identification accuracy.     

Reassessment of Species Diversity of the Subfamily Denticollinae (Coleoptera: Elateridae) through DNA Barcoding

The subfamily Denticollinae is a taxonomically diverse group in the family Elateridae. Denticollinae includes many morphologically similar species and crop pests, as well as many undescribed species at each local fauna. To construct a rapid and reliable identification system for this subfamily, the effectiveness of molecular species identification was assessed based on 421 cytochrome c oxidase subunit I (COI) sequences of 84 morphologically identified species. Among the 84 morphospecies, molecular species identification of 60 species (71.4%) was consistent with their morphological identifications. Six cryptic and/or pseudocryptic species with large genetic divergence (>5%) were confirmed by their sympatric or allopatric distributions. However, 18 species, including a subspecies, had ambiguous genetic distances and shared overlapping intra- and interspecific genetic distances (range: 2.12%–3.67%) suggesting incomplete lineage sorting, introgression of mitochondrial genome, or affection by endosymbionts, such as Wolbachia infection, between species and simple genetic variation within species. In this study, we propose a conservative threshold of 3.6% for convenient molecular operational taxonomic unit (MOTU) identification in the subfamily Denticollinae based on the results of pairwise genetic distances analyses using neighbor-joining, mothur, Automatic Barcode Gap Discovery analysis, and tree-based species delimitation by Poisson Tree Processes analysis. Using the 3.6% threshold, we identified 87 MOTUs and found 8 MOTUs in the interval between 2.5% to 3.5%. Evaluation of MOTUs identified in this range requires integrative species delimitation, including review of morphological and ecological differences as well as sensitive genetic markers. From this study, we confirmed that COI sequence is useful for reassessing species diversity for polymorphic and polytypic species occurring in sympatric and allopatric distributions, and for a single species having an extensively large habitat.     

ITS1 versus ITS2 as DNA metabarcodes for fungi

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut‐off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut‐off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under‐ or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.     

DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用

为了探讨DNA条形码技术在夜蛾物种鉴定中的可行性, 本研究利用条形码通用引物扩增了北京百花山地区43种夜蛾75个样本的线粒体细胞色素C氧化酶亚基I （mitochondrial cytochrome c oxidase subunit I, COI）基因序列, 以Kimura双参数模型进行种内种间遗传距离分析、 使用邻接法（neighbor-joining, NJ）和最大简约法（maximum parsimony, MP）分别构建系统发育树, 并利用分子序列差异阈值对样本进行分子可操作分类单元（molecular defined operational taxonomic units, MOTU）划分。结果表明： 所有夜蛾种类通过系统发育树可以成功区分; 种内平均遗传距离(0.03%)远远小于种间平均遗传距离(11.29%); 采用较为保守的1%的序列差异阈值将75个夜蛾样本分为42个MOTU, 正确率为95%, 除了MOTU04包含2个物种外, 剩余41个MOTU与形态种呈现一一对应的关系。结果显示, 基于COI基因的DNA条形码对于本研究中所涉及的夜蛾具有较好的区分, 可以作为一种有效的工具在夜蛾科昆虫物种鉴定中进行应用。     

Nutrient enrichment increased species richness of leaf litter fungal assemblages in a tropical forest

Microbial communities play a major role in terrestrial ecosystem functioning, but the determinates of their diversity and functional interactions are not well known. In this study, we explored leaf litter fungal diversity in a diverse Panama lowland tropical forest in which a replicated factorial N, P, K and micronutrient fertilization experiment of 40 × 40 m plots had been ongoing for nine years. We extracted DNA from leaf litter samples and used fungal‐specific amplification and a 454 pyrosequencing approach to sequence two loci, the nuclear ribosomal internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) D1 region. Using a 95% sequence similarity threshold for ITS1 spacer recovered a total of 2523 OTUs, and the number of unique ITS1 OTUs per 0.5–1.0 g leaf litter sample ranged from 55 to 177. Ascomycota were the dominant phylum among the leaf litter fungi (71% of the OTUs), followed by Basidiomycota (26% of the OTUs). In contrast to our expectations based on temperate ecosystems, long‐term addition of nutrients increased, rather than decreased, species richness relative to controls. Effect of individual nutrients was more subtle and seen primarily as changes in community compositions especially at lower taxonomic levels, rather than as significant changes in species richness. For example, plots receiving P tended to show a greater similarity in community composition compared to the other nutrient treatments, the +PK, +NK and +NPK plots appeared to be more dominated by the Nectriaceae than other treatments, and indicator species for particular nutrient combinations were identified.     

Barcoding the Collembola of Churchill: a molecular taxonomic reassessment of species diversity in a sub‐Arctic area

Although their functional importance in ecosystems is increasingly recognized, soil‐dwelling micro‐arthropods are usually poorly known in comparison with their above‐ground counterparts. Collembola constitute a significant and species‐rich component of the soil biodiversity, but it remains a woefully understudied group because of the taxonomic impediment. The ever‐increasing use of molecular taxonomic tools, such as DNA barcoding, provides a possible solution. Here, we test the use of this approach through a diversity survey of Collembola from the vicinity of Churchill, Manitoba, Canada, and compare the results with previous surveys in the same area and in other sub‐Arctic regions. The systematic barcoding campaign at Churchill revealed a diverse collembolan fauna consisting of 97 species‐level MOTUs in six types of habitats. If all these MOTUs are confirmed as species, this richness would be far higher than prior records for Arctic Canada and could lead to reconsider the actual diversity of the group in Arctic environments.     

Applying DNA barcoding for the study of geographical variation in host-parasitoid interactions

Studies on the biogeography of host-parasitoid interactions are scarce, mainly because of technical difficulties associated with rearing and species identification. DNA barcoding is increasingly recognized as a valuable tool for taxon identification, allowing to link different life history stages of a species. We evaluate the usefulness of a protocol based on cytochrome oxidase I (COI) sequencing for the study of geographical variation of host-parasitoid interactions. Larvae of Acroclita subsequana (Lepidoptera: Tortricidae) were collected in Macaronesia and dissected to search for parasitoid larvae. Both hosts and parasitoids were sequenced and assigned to molecular operational taxonomic units (MOTUs) based on pairwise genetic distances, tree-based and similarity-based methods. Hosts were grouped into six MOTUs, usually with an allopatric distribution, while parasitoids clustered into 12 MOTUs, each of which was mostly found attacking a single host MOTU. Available COI sequence databases failed to provide identification to species level for these MOTUs. Three challenges related to the applicability of DNA barcoding in this type of studies are identified and discussed: (i) more suitable primers need to be developed for both parasitoids and hosts; (ii) the most commonly used approaches for inferring MOTUs have different limitations (e.g. arbitrary nature of defining a threshold to separate MOTUs) and need to be improved or replaced by other techniques; and (iii) for the identification of MOTUs, it is imperative to increase the range of sequenced taxa in the currently available reference databases. Finally, in spite of these difficulties, we discuss how DNA barcoding will help ecological and biogeographical studies of host-parasitoid interactions.     

Patterns of Symbiodinium (Dinophyceae) diversity and assemblages among diverse hosts and the coral reef environment of Lizard Island,Australia

Large‐scale environmental disturbances may impact both partners in coral host–Symbiodinium systems. Elucidation of the assembly patterns in such complex and interdependent communities may enable better prediction of environmental impacts across coral reef ecosystems. In this study, we investigated how the community composition and diversity of dinoflagellate symbionts in the genus Symbiodinium were distributed among 12 host species from six taxonomic orders (Actinaria, Alcyonacea, Miliolida, Porifera, Rhizostoma, Scleractinia) and in the reef water and sediments at Lizard Island, Great Barrier Reef before the 3rd Global Coral Bleaching Event. 454 pyrosequencing of the ITS2 region of Symbiodinium yielded 83 operational taxonomic units (OTUs) at a 97% similarity cut‐off. Approximately half of the Symbiodinium OTUs from reef water or sediments were also present in symbio. OTUs belonged to six clades (A‐D, F‐G), but community structure was uneven. The two most abundant OTUs (100% matches to types C1 and A3) comprised 91% of reads and OTU C1 was shared by all species. However, sequence‐based analysis of these dominant OTUs revealed host species specificity, suggesting that genetic similarity cut‐offs of Symbiodinium ITS2 data sets need careful evaluation. Of the less abundant OTUs, roughly half occurred at only one site or in one species and the background Symbiodinium communities were distinct between individual samples. We conclude that sampling multiple host taxa with differing life history traits will be critical to fully understand the symbiont diversity of a given system and to predict coral ecosystem responses to environmental change and disturbance considering the differential stress response of the taxa within.     

Sequence clustering threshold has little effect on the recovery of microbial community structure

Analysis of microbial community structure by multivariate ordination methods, using data obtained by high‐throughput sequencing of amplified markers (i.e., DNA metabarcoding), often requires clustering of DNA sequences into operational taxonomic units (OTUs). Parameters for the clustering procedure tend not to be justified but are set by tradition rather than being based on explicit knowledge. In this study, we explore the extent to which ordination results are affected by variation in parameter settings for the clustering procedure. Amplicon sequence data from nine microbial community studies, representing different sampling designs, spatial scales and ecosystems, were subjected to clustering into OTUs at seven different similarity thresholds (clustering thresholds) ranging from 87% to 99% sequence similarity. The 63 data sets thus obtained were subjected to parallel DCA and GNMDS ordinations. The resulting community structures were highly similar across all clustering thresholds. We explain this pattern by the existence of strong ecological structuring gradients and phylogenetically diverse sets of abundant OTUs that are highly stable across clustering thresholds. Removing low‐abundance, rare OTUs had negligible effects on community patterns. Our results indicate that microbial data sets with a clear gradient structure are highly robust to choice of sequence clustering threshold.     

Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster

The bacteria in the fruitfly Drosophila melanogaster of different life stages was quantified by 454 pyrosequencing of 16S rRNA gene amplicons. The sequence reads were dominated by 5 operational taxonomic units (OTUs) at ≤ 97% sequence identity that could be assigned to Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, L. fructivorans and L. plantarum. The saturated rarefaction curves and species richness indices indicated that the sampling (85,000-159,000 reads per sample) was comprehensive. Parallel diagnostic PCR assays revealed only minor variation in the complement of the five bacterial species across individual insects and three D. melanogaster strains. Other gut-associated bacteria included 6 OTUs with low %ID to previously reported sequences, raising the possibility that they represent novel taxa within the genera Acetobacter and Lactobacillus. A developmental change in the most abundant species, from L. fructivorans in young adults to A. pomorum in aged adults was identified; changes in gut oxygen tension or immune system function might account for this effect. Host immune responses and disturbance may also contribute to the low bacterial diversity in the Drosophila gut habitat.     

Mitochondrial DNA (COI) analyses reveal that amphipod diversity is associated with environmental heterogeneity in deep‐sea habitats

The relationship between species diversity and environmental parameters is poorly understood for the mobile macrofauna of deep‐sea habitats due to under‐sampling and subsequent lack of accurate taxonomic information. To redress this, cytochrome oxidase c subunit I (COI) DNA sequences were used to estimate species diversity and to compare phoxocephalid amphipod assemblages among 20 stations encompassing a range of environmental conditions. Two regions, east (Chatham Rise) and west (Challenger Plateau) of New Zealand were sampled to depths of 200–1200 m with an epibenthic sled. Using a comparison among identified morphospecies, we found a clear gap in sequence divergences between 6% and 13% and used a 6% threshold to designate molecular operational taxonomic units (MOTUs), as a surrogate to putative species. DNA sequences (n = 297) revealed high total diversity (n = 49 MOTUs), as well as high beta diversity (28 MOTUs found at single location only). Novel phoxocephalid MOTUs were found at most stations, especially on Challenger Plateau and the flanks of Chatham Rise. Analyses of interstation assemblages revealed a major split between regions, indicating minimal overlap in taxon distributions. A cluster of highly similar stations was identified, broadly distributed over the crest of Chatham Rise, in association with elevated food availability, probably resulting from higher surface productivity and relatively shallow depth. Accordingly, multivariate analysis revealed a strong correlation between phoxocephalid assemblages and food supply. This study highlights the value of molecular approaches, in particular COI sequences, for quantifying and comparing diversity in under‐sampled and/or under‐studied taxa.     

Ironing out the wrinkles in the rare biosphere through improved OTU clustering

Deep sequencing of PCR amplicon libraries facilitates the detection of low‐abundance populations in environmental DNA surveys of complex microbial communities. At the same time, deep sequencing can lead to overestimates of microbial diversity through the generation of low‐frequency, error‐prone reads. Even with sequencing error rates below 0.005 per nucleotide position, the common method of generating operational taxonomic units (OTUs) by multiple sequence alignment and complete‐linkage clustering significantly increases the number of predicted OTUs and inflates richness estimates. We show that a 2% single‐linkage preclustering methodology followed by an average‐linkage clustering based on pairwise alignments more accurately predicts expected OTUs in both single and pooled template preparations of known taxonomic composition. This new clustering method can reduce the OTU richness in environmental samples by as much as 30–60% but does not reduce the fraction of OTUs in long‐tailed rank abundance curves that defines the rare biosphere.