Species abundance distributions and richness estimations in fungal metagenomics--lessons learned from community ecology

Results of diversity and community ecology studies strongly depend on sampling depth. Completely surveyed communities follow log-normal distribution, whereas power law functions best describe incompletely censused communities. It is arguable whether the statistics behind those theories can be applied to voluminous next generation sequencing data in microbiology by treating individual DNA sequences as counts of molecular taxonomic units (MOTUs). This study addresses the suitability of species abundance models in three groups of plant-associated fungal communities - phyllosphere, ectomycorrhizal and arbuscular mycorrhizal fungi. We tested the impact of differential treatment of molecular singletons on observed and estimated species richness and species abundance distribution models. The arbuscular mycorrhizal community of 48 MOTUs was exhaustively sampled and followed log-normal distribution. The ectomycorrhizal (153 MOTUs) and phyllosphere (327 MOTUs) communities significantly differed from log-normal distribution. The fungal phyllosphere community in particular was clearly undersampled. This undersampling bias resulted in strong sensitivity to the exclusion of molecular singletons and other rare MOTUs that may represent technical artefacts. The analysis of abundant (core) and rare (satellite) MOTUs clearly identified two species abundance distributions in the phyllosphere data - a log-normal model for the core group and a log-series model for the satellite group. The prominent log-series distribution of satellite phyllosphere fungi highlighted the ecological significance of an infrequent fungal component in the phyllosphere community.     

Blind assessment of vertebrate taxonomic diversity across spatial scales by clustering environmental DNA metabarcoding sequences

Human activities impact all ecosystems on Earth, which urges scientists to better understand biodiversity changes across temporal and spatial scales. Environmental DNA (eDNA) metabarcoding is a promising non-invasive method to assess species composition in a wide range of ecosystems. Yet, this method requires the completeness of a reference database, i.e. a list of DNA sequences attached to each species of the regional pool, which is rarely met. As an alternative, molecular operational taxonomic units (MOTUs) can be extracted as clusters of sequences. However, the extent to which the diversity of MOTUs can predict the diversity of species across spatial scales is unknown. Here, we used 196 samples along the Rhone river (France) for which the reference database is complete to assess whether a blind eDNA approach can reliably predict the ground-truth number of species at different spatial scales. Using the 12S rDNA teleo primer, we curated and clustered 60 million sequences into MOTUs using a new assembled bioinformatic pipeline. We show that stringent quality filters were necessary to remove artefact noise, notably MOTUs present in a single PCR replicate, which represented 55% of MOTUs (103). Post-clustering cleaning also removed 19 additional erroneous MOTUs and only discarded one truly present species. We then show that the diversity of retained fish MOTUs accurately predicted the local (α, r = 0.98) and regional (γ) ground-truth species diversity (67 MOTUs versus 63 species), but also the species dissimilarity between samples (β-diversity, r = 0.98). This work paves the way towards extending the use of eDNA metabarcoding in community ecology and biogeography despite major gaps in genetic reference databases.     

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.     

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.     

A metagenetic approach for revealing community structure of marine planktonic copepods

Marine planktonic copepods are an ecologically important group with high species richness and abundance. Here, we propose a new metagenetic approach for revealing the community structure of marine planktonic copepods using 454 pyrosequencing of nuclear large subunit ribosomal DNA. We determined an appropriate similarity threshold for clustering pyrosequencing data into molecular operational taxonomic units (MOTUs) using an artificial community containing 33 morphologically identified species. The 99% similarity threshold had high species‐level resolution for MOTU clustering but overestimated species richness. The artificial community was appropriately clustered into MOTUs at 97% similarity, with little inflation in MOTU numbers and with relatively high species‐level resolution. The number of sequence reads of each MOTU was correlated with dry weight of that taxon, suggesting that sequence reads could be used as a proxy for biomass. Next, we applied the method to field‐collected samples, and the results corresponded reasonably well with morphological analysis of these communities. Numbers of MOTUs were well correlated with species richness at 97% similarity, and large numbers of sequence reads were generally observed in MOTUs derived from species with large biomass. Further, MOTUs were successfully classified into taxonomic groups at the family level at 97% similarity; similar patterns of species richness and biomass were revealed within families with metagenetic and morphological analyses. At the 99% similarity threshold, MOTUs with high proportions of sequence reads were identified as biomass‐dominant species in each field‐collected sample. The metagenetic approach reported here can be an effective tool for rapid and comprehensive assessment of copepod community structure.     

Microfungi associated with withering willow wood in ground contact near Syowa Station, East Antarctica for 40 years

Data are rather lacking on the diversity of microfungi associated with exotic plant substrates transported to continental Antarctica. We examined the diversity and species composition of microfungi associated with withering woody shoots of saplings of Salix spp. (willows) transplanted and in ground contact near Syowa Station, East Antarctica for more than 40 years. The willow saplings originated from Hokkaido, Northern Japan, and were experimentally transplanted in 1967–1968, but died within a few years. Dead willow shoots, unbranched and standing on bare ground for approximately 50 years, were used for the isolation of fungi with the surface disinfection method. A total of 43 isolates were retrieved from 32 (78 %) of the 41 shoots tested. The fungal isolates were classified into 18 molecular operational taxonomic units (MOTUs) based on the similarity of rDNA ITS sequences at the 97 % criterion. Leotiomycetes was the most common class in terms of the number of isolates and MOTUs, followed by Dothideomycetes, Sordariomycetes, and Eurotiomycetes. Molecular phylogenetic affinities suggested that the closest relatives of the MOTUs were saprobic and root-associated fungi. The result of the present study suggested that Cadophora luteo-olivacea is widespread in soils throughout Antarctica and likely indigenous.     

Mitochondrial divergence suggests unexpected high species diversity in the opsariichthine fishes (Teleostei: Cyprinidae) and the revalidation of Opsariichthys macrolepis

Opsariichthine (sensu Oceanologi Et Limnologia Sinica, 1982, 13, 293–298) is a cyprinid group consisting of five genera and endemic to East Asia. Previous studies suggested that there may be many possible cryptic species in this group, but this has not been confirmed. In this study, using mitochondrial cyt b sequences on 1,388 samples and 739 haplotypes, we showed very high species diversity within this group. The results showed that phylogenetic relationships of the opsariichthine group were as ([Nipponocypris‐Parazacco‐Candidia] + [Zacco + Opsariichthys]), and there were multiple deep lineages within several species, flagging putative cryptic species. When a 3% genetic distance was used as a threshold for species delimitation, 35 haplogroups were found, nine haplogroups in Candidia‐Parazacco‐Nipponocypris group, six haplogroups in the Zacco group, and 20 haplogroups in the Opsariichthys group. We consider all of them to be putative until determination of distinct species based on the tree topology, geographic distributions, or a combination of both. In addition, two kinds of species delimitation tools, ABGD and PTP, were applied to construct molecular operational taxonomic units (MOTUs). The ABGD method revealed nine MOTUs in Candidia‐Parazacco‐Nipponocypris group, two MOTUs in the Zacco group, and 17 MOTUs in the Opsariichthys group. And the PTP method revealed 10 MOTUs in Candidia‐Parazacco‐Nipponocypris group, 10 MOTUs in the Zacco group, and 29 MOTUs in the Opsariichthys group. Therefore, there should be more species in the opsariichthine group than presently described. Based on the molecular data and morphological characteristics, we proposed Opsariichthys macrolepis as a valid species and described its morphological diagnostic characters.     

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.     

Trade-offs in community ecology: linking spatial scales and species coexistence

Trade‐offs in species performances of different ecological functions is one of the most common explanations for coexistence in communities. Despite the potential for species coexistence occurring at local or regional spatial scales, trade‐offs are typically approached at a single scale. In recent years, ecologists have increasingly provided evidence for the importance of community processes at both local and regional spatial scales. This review summarizes the theoretical predictions for the traits associated with trade‐offs under different conditions and at different spatial scales. We provide a spatial framework for understanding trade‐offs, coexistence and the supportive empirical evidence. Predictions are presented that link the patterns of diversity observed to the patterns of trade‐offs that lead to coexistence at different spatial scales. Recent evidence for the evolution of trade‐offs under different conditions is provided which explores both laboratory microcosm studies and phylogenetic tests. Examining trade‐offs within a spatial framework can provide a strong approach to understanding community structure and dynamics, while explaining patterns of species diversity.     

Cobble community DNA as a tool to monitor patterns of biodiversity within kelp forest ecosystems

Kelp forest ecosystems dominate 150,000 km of global temperate coastline, rivalling the coastal occurrence of coral reefs. Despite the astounding biological diversity and productive ecological communities associated with kelp forests, patterns of species richness and composition are difficult to monitor and compare. Crustose coralline algae are a critically important substrate for propagule settlement for a range of kelp forest species. Coralline‐covered cobbles are home to hundreds of species of benthic animals and algae and form a replicable unit for ecological assays. Here, we use DNA metabarcoding of bulk DNA extracts sampled from cobbles to explore patterns of species diversity in kelp forests of the central California coast. The data from 97 cobbles within kelp forest ecosystems at three sites in Central California show the presence of 752 molecular operational taxonomic units (MOTUs) and 53 MOTUs assigned up to the species level with >95% similarity to current databases. We are able to detect spatial patterns of important management targets such as abalone recruits, and localized abundance of sea stars in 2012. Comparison of classic ecological surveys of these sites reveals large differences in species targets for these two approaches. In order to make such comparisons more quantitative, we use Presence/Absence Metabarcoding, using the fraction of replicate cobbles showing a species as a measure of its local abundance. This approach provides a fast and repeatable survey method that can be applied for biodiversity assessments across systems to shed light on the impact of different ecological disturbances and the role played by marine protected areas.     

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.     

Confrontation of cryptic diversity and mate discrimination within Gammarus pulex and Gammarus fossarum species complexes

相似文献   

Where is the unseen fungal diversity hidden? A study of Mortierella reveals a large contribution of reference collections to the identification of fungal environmental sequences

? Estimation of the proportion of undescribed fungal taxa is an issue that has remained unresolved for many decades. Several very different estimates have been published, and the relative contributions of traditional taxonomic and next-generation sequencing (NGS) techniques to species discovery have also been called into question recently. ? Here, we addressed the question of what proportion of hitherto unidentifiable molecular operational taxonomic units (MOTUs) have already been described but not sequenced, and how many of them represent truly undescribed lineages. We accomplished this by modeling the effects of increasing type strain sequencing effort on the number of identifiable MOTUs of the widespread soil fungus Mortierella. ? We found a nearly linear relationship between the number of type strains sequenced and the number of identifiable MOTUs. Using this relationship, we made predictions about the total number of Mortierella species and found that it was very close to the number of described species in Mortierella. ? These results suggest that the unusually high number of unidentifiable MOTUs in environmental sequencing projects can be, at least in some fungal groups, ascribed to a lag in type strain and specimen sequencing rather than to a high number of undescribed species.     

Evolutionary criteria outperform operational approaches in producing ecologically relevant fungal species inventories

Analyses of the structure and function of microbial communities are highly constrained by the diversity of organisms present within most environmental samples. A common approach is to rely almost entirely on DNA sequence data for estimates of microbial diversity, but to date there is no objective method of clustering sequences into groups that is grounded in evolutionary theory of what constitutes a biological lineage. The general mixed Yule-coalescent (GMYC) model uses a likelihood-based approach to distinguish population-level processes within lineages from processes associated with speciation and extinction, thus identifying a distinct point where extant lineages became independent. Using two independent surveys of DNA sequences associated with a group of ubiquitous plant-symbiotic fungi, we compared estimates of species richness derived using the GMYC model to those based on operational taxonomic units (OTUs) defined by fixed levels of sequence similarity. The model predicted lower species richness in these surveys than did traditional methods of sequence similarity. Here, we show for the first time that groups delineated by the GMYC model better explained variation in the distribution of fungi in relation to putative niche-based variables associated with host species identity, edaphic factors, and aspects of how the sampled ecosystems were managed. Our results suggest the coalescent-based GMYC model successfully groups environmental sequences of fungi into clusters that are ecologically more meaningful than more arbitrary approaches for estimating species richness.     

Application of DNA barcoding in biodiversity studies of shallow-water octocorals: molecular proxies agree with morphological estimates of species richness in Palau

The application of DNA barcoding to anthozoan cnidarians has been hindered by their slow rates of mitochondrial gene evolution and the failure to identify alternative molecular markers that distinguish species reliably. Among octocorals, however, multilocus barcodes can distinguish up to 70 % of morphospecies, thereby facilitating the identification of species that are ecologically important but still very poorly known taxonomically. We tested the ability of these imperfect DNA barcodes to estimate species richness in a biodiversity survey of the shallow-water octocoral fauna of Palau using multilocus (COI, mtMutS, 28S rDNA) sequences obtained from 305 specimens representing 38 genera of octocorals. Numbers and identities of species were estimated independently (1) by a taxonomic expert using morphological criteria and (2) by assigning sequences to molecular operational taxonomic units (MOTUs) using predefined genetic distance thresholds. Estimated numbers of MOTUs ranged from 73 to 128 depending on the barcode and distance threshold applied, bracketing the estimated number of 118 morphospecies. Concordance between morphospecies identifications and MOTUs ranged from 71 to 75 % and differed little among barcodes. For the speciose and ecologically dominant genus Sinularia, however, we were able to identify 95 % of specimens correctly simply by comparing mtMutS sequences and in situ photographs of colonies to an existing vouchered database. Because we lack a clear understanding of species boundaries in most of these taxa, numbers of morphospecies and MOTUs are both estimates of the true species diversity, and we cannot currently determine which is more accurate. Our results suggest, however, that the two methods provide comparable estimates of species richness for shallow-water Indo-Pacific octocorals. Use of molecular barcodes in biodiversity surveys will facilitate comparisons of species richness and composition among localities and over time, data that do not currently exist for any octocoral community.     

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

相似文献   

Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units

The lack of a robust taxonomy in the genus Saprolegnia is leading to the presence of incorrectly named isolates in culture collections and of an increasing number of misassigned sequences in DNA databases. Accurate species delimitation is critical for most biological disciplines. A recently proposed approach to solve species delimitation (taxonomic diagnosis system) of difficult organisms is the definition of molecular operational taxonomic units (MOTUs). We have used 961 sequences of nrDNA ITS from culture collections (461 sequences) and GenBank (500 sequences), to perform phylogenetic and clustering optimization analyses. As result, we have identified 29 DNA-based MOTUs in agreement with phylogenetic studies. The resulting molecular clusters support the validity of 18 species of Saprolegnia and identify 11 potential new ones. We have also listed a number of incorrectly named isolates in culture collections, misassigned species names to GenBank sequences, and reference sequences for the species. We conclude that GenBank represents the main source of errors for identifying Saprolegnia species since it possesses sequences with misassigned names and also sequencing errors. The presented taxonomic diagnosis system might help setting the basis for a suitable identification of species in this economically important genus.     

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.     

Macaronesian islands as promoters of diversification in amphipods: The remarkable case of the family Hyalidae (Crustacea,Amphipoda)

The north‐east Atlantic (NEA) is an important and complex biogeographic region with a very rich marine fauna. However, little is known about the role of the Macaronesian islands in the evolutionary history and diversification of marine invertebrates in the NEA. Among the amphipods, the members of the family Hyalidae are particularly common and abundant in intertidal rocky shores of NEA. In this study, we aimed to investigate the genetic structure and diversity of seven hyalid species inhabiting the Macaronesian, European and Moroccan Atlantic coasts, with a focus on the genetic differentiation between island and Continental populations. Analysis of mitochondrial DNA sequences of the cytochrome oxidase I gene, unravelled a very high level of hidden diversity, consisting of 26–32 molecular operational taxonomic units (MOTUs), the majority of them recorded in Macaronesian populations. Except for Apohyale stebbingi, all remaining MOTUs were in general allopatrically distributed, with a trend for segregation between islands and Continental populations on one side, but also for the occurrence of private MOTUs among islands. Results indicate distinct evolutionary and diversification patterns among Hyalidae species, but a strong separation between Continental and islands’ lineages appears to be a common feature to all of them. Apparently, the complex geomorphological history of the Macaronesian archipelagos served as an important promoter of extensive diversification of marine invertebrates in NEA, a phenomenon which only now starts to be fully appreciated through the use of molecular data.     

One‐locus‐several‐primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets’ complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the “one‐locus‐several‐primer‐sets” (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey–predator interactions.