The promise of a DNA taxonomy

Not only is the number of described species a very small proportion of the estimated extant number of taxa, but it also appears that all concepts of the extent and boundaries of 'species' fail in many cases. Using conserved molecular sequences it is possible to define and diagnose molecular operational taxonomic units (MOTU) that have a similar extent to traditional 'species'. Use of a MOTU system not only allows the rapid and effective identification of most taxa, including those not encountered before, but also allows investigation of the evolution of patterns of diversity. A MOTU approach is not without problems, particularly in the area of deciding what level of molecular difference defines a biologically relevant taxon, but has many benefits. Molecular data are extremely well suited to re-analysis and meta-analysis, and data from multiple independent studies can be readily collated and investigated by using new parameters and assumptions. Previous molecular taxonomic efforts have focused narrowly. Advances in high-throughput sequencing methodologies, however, place the idea of a universal, multi-locus molecular barcoding system in the realm of the possible.     

Molecular barcodes for soil nematode identification

Using a molecular barcode, derived from single-specimen polymerase chain reaction (PCR) and sequencing of the 5' segment of the small subunit ribosomal RNA (SSU) gene, we have developed a molecular operational taxonomic unit (MOTU) scheme for soil nematodes. Individual specimens were considered to belong to the same MOTU when the sequenced segment of 450 bases was > 99.5% identical. A Scottish upland Agrostis-Festuca grassland soil was sampled, using both culture-based and random selection methods. One hundred and sixty-six cultured isolates were sequenced, and clustered into five MOTU. From 74 randomly sampled individuals across the study site, 19 MOTU were defined. A subsequent sample of 18 individuals from a single subplot contained eight MOTU, four of which were unique to the single subplot sample. Interestingly, seven of these MOTU were not present in the culture-independent sampling. Overall, a total of 23 MOTU were defined from only 240 sequences. Many MOTU could readily be assigned to classical, morphologically defined taxonomic units using a database of SSU sequences from named nematode species. The MOTU technique allows a rapid assessment of nematode taxon diversity in soils. Correlation with a database of sequences from known species offers a route to application of the technique in ecological surveys addressing biological as well as genetic diversity.     

At least some meiofaunal species are not everywhere. Indication of geographic,ecological and geological barriers affecting the dispersion of species of Ototyphlonemertes (Nemertea,Hoplonemertea)

Most meiofaunal species are known to have a broad distribution with no apparent barriers to their dispersion. However, different morphological and/or molecular methods supported patterns of diversity and distribution that may be different among taxa while also conflicting within the same group. We accurately assessed the patterns of geographic distribution in actual genetic species of a marine meiofaunal animal model: Ototyphlonemertes. Specimens were collected from several sites around Europe, Northern and Central America, Southern America, Pacific Islands and Asia. We sequenced regions of two mitochondrial and two nuclear genes. Using single‐gene, a concatenated data set, multilocus approaches and different DNA taxonomy methods, we disentangled the actual diversity and the spatial structures of haplotypes and tested the possible correlation between genetic diversity and geographic distance. The results show (i) the importance of using several genes to uncover both diversity and highlight phylogeographic relationships among species and that (ii) independent genetic evolutionary entities have a narrower distribution than morphological species. Moreover, (iii) a Mantel test supported a positive correlation between genetic and geographical distance. By sampling from the two sides of Isthmus of Panama, we were additionally able to identify lineage divergence times that are concordant with vicariance mechanisms caused by the geological closure of the seaway across the Isthmus. We therefore propose that in addition to distance, other geological and ecological conditions are also barriers to the dispersion of and gene flow in marine meiofaunal organisms.     

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

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The effects of nutrient enrichment on a freshwater meiofaunal assemblage

1. The effects of eutrophication on phytoplankton, zooplankton and fish in lakes are well known. By contrast, little is known about the response of the zoobenthos to nutrient enrichment, while smaller organisms, such as the meiofauna, have for the most part been neglected. 2. In a long‐term (16 months) microcosm experiment, we assessed the effects of five levels of nutrients [total phosphorus (TP), 7–250 μg L^?1; nitrate, 2–8 mg L^?1] on a freshwater meiofaunal assemblage and on nematode diversity in particular. 3. Within the first 8 months, meiofaunal succession was only weakly affected, whereas, during the last 4 months, nutrient addition influenced most of the main taxa, with a concomitant change in the assemblage structure. 4. The density of the numerically dominant nematodes decreased upon nutrient enrichment, whereas ostracods became more numerous. Other taxa, including copepods, reached a maximum at intermediate nutrient levels or, in case of oligochaetes, were almost unaffected by nutrient enrichment. However, the changes in the density of the main taxa were usually insufficient to alter their biomass. Consequently, meiofaunal biomass was remarkably unresponsive to nutrient addition, while meiofaunal density displayed a unimodal relationship, with a peak at a TP concentration of 30 μg L^?1. In addition, nematode species richness decreased significantly with increasing nutrient concentrations. 5. We hypothesise that the response of meiofaunal taxa to nutrients is attributable to the development of primary producers, which shifted with enrichment from low densities of edible diatoms and unicellular green algae to large standing stocks of inedible forms, such as Lemna minor and Cladophora spp.     

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.     

jMOTU and Taxonerator: turning DNA Barcode sequences into annotated operational taxonomic units

Background

DNA barcoding and other DNA sequence-based techniques for investigating and estimating biodiversity require explicit methods for associating individual sequences with taxa, as it is at the taxon level that biodiversity is assessed. For many projects, the bioinformatic analyses required pose problems for laboratories whose prime expertise is not in bioinformatics. User-friendly tools are required for both clustering sequences into molecular operational taxonomic units (MOTU) and for associating these MOTU with known organismal taxonomies.

Results

Here we present jMOTU, a Java program for the analysis of DNA barcode datasets that uses an explicit, determinate algorithm to define MOTU. We demonstrate its usefulness for both individual specimen-based Sanger sequencing surveys and bulk-environment metagenetic surveys using long-read next-generation sequencing data. jMOTU is driven through a graphical user interface, and can analyse tens of thousands of sequences in a short time on a desktop computer. A companion program, Taxonerator, that adds traditional taxonomic annotation to MOTU, is also presented. Clustering and taxonomic annotation data are stored in a relational database, and are thus amenable to subsequent data mining and web presentation.

Conclusions

jMOTU efficiently and robustly identifies the molecular taxa present in survey datasets, and Taxonerator decorates the MOTU with putative identifications. jMOTU and Taxonerator are freely available from http://www.nematodes.org/.     

The distribution of coastal fish eDNA sequences in the Anthropocene

Aim

Coastal fishes have a fundamental role in marine ecosystem functioning and contributions to people, but face increasing threats due to climate change, habitat degradation and overexploitation. The extent to which human pressures are impacting coastal fish biodiversity in comparison with geographic and environmental factors at large spatial scale is still under scrutiny. Here, we took advantage of environmental DNA (eDNA) metabarcoding to investigate the relationship between fish biodiversity, including taxonomic and genetic components, and environmental but also socio-economic factors.

Location

Tropical, temperate and polar coastal areas.

Time period

Present day.

Major taxa studied

Marine fishes.

Methods

We analysed fish eDNA in 263 stations (samples) in 68 sites distributed across polar, temperate and tropical regions. We modelled the effect of environmental, geographic and socio-economic factors on α- and β-diversity. We then computed the partial effect of each factor on several fish biodiversity components using taxonomic molecular units (MOTU) and genetic sequences. We also investigated the relationship between fish genetic α- and β-diversity measured from our barcodes, and phylogenetic but also functional diversity.

Results

We show that fish eDNA MOTU and sequence α- and β-diversity have the strongest correlation with environmental factors on coastal ecosystems worldwide. However, our models also reveal a negative correlation between biodiversity and human dependence on marine ecosystems. In areas with high dependence, diversity of all fish, cryptobenthic fish and large fish MOTUs declined steeply. Finally, we show that a sequence diversity index, accounting for genetic distance between pairs of MOTUs, within and between communities, is a reliable proxy of phylogenetic and functional diversity.

Main conclusions

Together, our results demonstrate that short eDNA sequences can be used to assess climate and direct human impacts on marine biodiversity at large scale in the Anthropocene and can further be extended to investigate biodiversity in its phylogenetic and functional dimensions.     

Diversity and phylogenetic community structure of ants along a Costa Rican elevational gradient

The diversity and phylogenetic community structure of many organisms is negatively affected by factors that covary with elevation. On the Pacific slope of the Cordillera Guanacaste within Area de Conservación Guanacaste (ACG) in northwestern Costa Rica we found a negative relationship between elevation and ant diversity on each of three volcanos. This pattern was evident when diversity was measured through molecular operational taxonomic units (MOTU) or by phylogenetic diversity (PD) based on DNA barcodes or a multi‐gene phylogeny. We observed an asymmetrical mid‐elevation peak at approximately 600–800 m and we found high species turnover between sites on the same mountain and among the three mountains. At the highest elevation cloud forest sites we found evidence of significant phylogenetic clustering, the expected result of environmental filtering. The narrow elevational range of each species, coupled with the high diversity at each sampling point, emphasizes that climate change will bring strong changes in the location and composition of biodiversity on these mountains. The structure and composition of the hyperdiverse communities present at any one elevation is extremely vulnerable to a changing climate.     

Assessment of ectomycorrhizal biodiversity in Tuber macrosporum productive sites

Tuber macrosporum Vittad. is a truffle with superb organoleptic properties, whose cultivation is still in its infancy. For the first time we have aimed to provide information on ectomycorrhizal communities in natural and cultivated T. macrosporum sites. Ectomycorrhizal morphotypes were identified using ITS nrDNA sequencing and sorted into molecular operational taxonomic unit (MOTU). We detected 16 MOTUs in the T. macrosporum cultivated plantation. Ascomycota were the most abundant (86.4 %) with Helvellaceae, Pyronemataceae and Pezizaceae the most common. Twenty-two MOTUs were collected in the natural T. macrosporum site. Basidiomycota morphotypes were plentiful (70.6 %) and Thelephoraceae dominated. Each site had different taxa belowground with only T. macrosporum in common, being more abundant in the natural (18.2 %) than in the cultivated (14.4 %) site. Species richness, Simpson and Shannon diversity indices, taxonomic diversity, distinctness and variation of taxonomic distinctness were lower in the cultivated than in the natural site.     

The use of meiofauna in freshwater sediment assessments: Structural and functional responses of meiobenthic communities to metal and organics contamination

Because soft sediments are often hotspots of chemical contamination, their assessment can aid in identifying the causes of environmental stress and the implementation of measures to improve the health of the respective ecosystems. Achieving a “good ecological status” of surface waters, as required by the European Water Framework Directive, strongly depends on recognition of the chemical status of sediments. Meiobenthic organisms are important, but widely neglected components of the ecologically relevant fauna of a wide variety of ecosystems. In the present study, microcosms containing freshwater sediments were used to investigate the effects of eight different metals and polycyclic aromatic hydrocarbons (PAHs), in single and mixed applications, on natural meiofaunal assemblages. Structural (abundance and biomass) and functional (secondary production) parameters of the investigated assemblages were measured as ecologically relevant endpoints. Their sensitivity in revealing both the differential effects and the responses of meiofaunal taxa was evaluated to assess the general suitability of meiofauna and, in particular, of individual tested taxa, as bioindicators of soft sediment contamination. Structural parameters were found to be more valuable indicators than functional measurements, with more pronounced effects observed on the taxon level than on total meiofauna. Among the meiofaunal taxa considered in this study, nematodes were of particular utility as early indicators of chemical stress in freshwater soft sediments. Overall, this study provides new insights into the impact of toxicants on soft freshwater sediments and demonstrates the suitability of meiofaunal communities, especially nematodes, in assessing contamination of this ecosystem.     

北京市区苔藓植物空间分布与环境关系的研究

在2006年10月至2007年10月期间,对北京市五环路内及附近部分区域的苔藓群落进行了调查,共调查了31个地点、164个样地,采集苔藓植物标本327份。经整理鉴定,调查区有苔藓植物12科24属55种,其中阔叶小石藓（Weisia planifolia）和立碗藓（Physcomitrium sphaericum）等种类为调查区的广布种。通过对调查区同类型、不同地点苔藓植物多样性指数分析,发现北京市苔藓植物的分布特点为城西、城北苔藓植物多样性高于城东、城南。用典范对应分析法（CCA）分析27个样地中的20种地面生苔藓植物的盖度与主要环境因子的关系,结果表明,土壤含水量、距主干道距离、人为踩踏、土壤pH值、林木郁闭度等环境因子对北京市苔藓植物的分布有显著影响,其中土壤含水量、土壤pH值对苔藓植物分布的影响最为显著,北京市区大多数常见苔藓种类喜生于土壤含水量较高、偏酸性的环境。     

The importance of turbellarians in the marine meiobenthos: a review

Recent data and opinions on meiofaunal ecology are briefly reviewed; and from scattered data, the place of turbellarians in the meiobenthic community is discussed. Turbellarian diversity, density, and biomass are higher in sandy habitats than in muddy bottoms. In sand, turbellarian diversity is of the same magnitude as that of other important meiofaunal taxa, while densities range between 7–25% of the total meiofauna. Mean individual turbellarian dry weight seems to be four times that of nematodes and in sandy habitats turbellarian biomass may be equal to or excede that of nematodes. Most turbellarian species may be considered as predators and in this respect may take the place occupied by macrofaunal species in muddy sediments. mens.

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Experimental evidence of predation by juvenile flounder, Platichthys flesus, on a shallow water meiobenthic community

In the northern Baltic Sea juvenile flounder, Platichthys flesus, occur in high abundances on shallow sandy bottoms in late summer and autumn. They feed mainly on meiofauna and the ontogenetic switch to macrofaunal sized food occurs at a larger size here than in other areas, exemplifying the high relative importance of meiofauna. Consequently, juvenile P. flesus in the Baltic feed for a longer period on meiofauna, and could thus be expected to have a stronger predation effect on the meiofaunal assemblages. In this study the predation effects of juvenile P. flesus on meiofaunal abundances and community structure were studied using microcosms that were sampled repeatedly over a 3-week period. Significant differences between treatment and control were found for the total number of taxa, for abundances of harpacticoids, copepod nauplii and ostracods. The nematode community was not affected, but one genus, Axonolaimus, was negatively affected by predation. The predation affected meiofaunal community structure as the major taxon diversity was significantly reduced. The results suggest that the meiofauna on shallow sandy bottoms may be structured by juvenile P. flesus, and the combined predation pressure of juvenile flounder and other epibenthic predators in the area might be considerable. Microcosms are effective in testing natural predation, especially on meiofaunal communities, but field experiments should be conducted to account for the physical characteristics of the area studied.     

The meiofaunal community structure of a Mediterranean lagoon (Gialova lagoon, Ionian Sea)

The higher taxonomic structure of a meiofaunal community was investigated in an Eastern Mediterranean lagoon (Gialova lagoon, Ionian Sea). Seven stations were sampled on a seasonal basis during 1995–1996. Stations within the lagoon were found to support a maximum of 14 meiofaunal taxa with densities ranging from 17 to over 2000 individuals per 10 cm². Nematodes were not always the most abundant taxa, although they dominated about half of the total 28 samples. Uni- and multivariate analyses were employed to study the community structure. The distribution pattern of the meiofaunal community varied both across the lagoon and over the seasons. On the basis of the spatial differences a meiofaunal coenocline, correlated with the degree of isolation, was observed, composed of mainly two zones: one defined by the area close to the marine channel and the other the more isolated area in the inner lagoon. Meiofaunal distribution pattern was not clearly correlated to one environmental variable, but rather to many, and spatial and seasonal effects could be seen. Received: 2 March 1999 / Received in revised form: 6 December 1999 / Accepted: 9 December 1999     

THE POTENTIAL INDICATOR VALUE OF RARE TAXA RICHNESS IN DIATOM‐BASED STREAM BIOASSESSMENT1

Despite their recognized contribution to species richness, the importance of rare taxa richness in bioassessment is unclear. This study aimed to characterize the environmental factors affecting the number of rare diatom taxa in western U.S. streams and rivers, and to evaluate whether this number can be used to differentiate streams with contrasting human disturbance. Three different categories of rare taxa were used: satellite (taxa with low occurrence and low abundance), rural (taxa with high occurrence and low abundance), and urban (taxa with low occurrence and high abundance). Common taxa were included as a separate category of core taxa (taxa with high occurrence and high abundance). We analyzed 987 diatom samples collected over the period of 5 years (2000–2004) for the U.S. Environmental Protection Agency’s (EPA) Western Environmental Monitoring and Assessment Program (WEMAP). The results showed that rural taxa richness (number of rural taxa per site) increased along a longitudinal gradient from mountainous, fast‐flowing oligotrophic streams with fewer fine substrates to large, slow‐moving, nutrient‐rich rivers with abundance of fine substrates. Rural taxa richness was the only rarity metric that distinguished least disturbed (reference) sites from the most disturbed (impacted) sites, but it was significantly different only in the mountains ecoregion. Core taxa richness distinguished reference from impacted sites in the West and in each one of the three ecoregions (mountains, plains, and xeric). Our findings revealed that rural taxa richness can be used as an indicator of human disturbance in streams/rivers, especially in the mountains ecoregion, and that rarity definition is important in bioassessment.     

Synoptic patterns of meiofaunal and macrofaunal abundances and specific composition in littoral sediments

During recent years, many investigations on small zoobenthos have been performed at the island of Sylt. As these studies were carried out sporadically over many years and as different extraction methods were used, comparisons of the results have been hampered. Therefore, in August/September 1986, 24 sites were sampled and evaluated using one quantitative method throughout. Sites range from mud to exposed sand and from the sublittoral to the supralittoral. Macrofauna and the taxa Plathelminthes, Polychaeta, and Oligochaeta are determined to species level. Macrofaunal (>0.5 mm) abundance is highest in mud and continuously decreases with increasing exposure to wave action. Meiofaunal (<0.5 mm) abundance is less variable. Nematoda dominate in mud and muddy sand, Copepoda in sheltered and exposed sand, other taxa only intermittently. Related to surface area, no correlation between macro-and meiofaunal abundance is apparent. Plathelminthes and Copepoda reach highest abundance per surface area in sand but their per volume density is higher in mud and muddy sand. Related to sediment volume instead of surface area, the meiofaunal abundance pattern is very similar to the macrofaunal pattern. The faunal composition changes gradually along the tidal gradient without general faunal boundaries. On an averange, the faunal similarity of neighbouring sites is highest in Oligochaeta and lowest in Plathelminthes. Presumably, Oligochaeta tolerate wider ranges of environmental factors. This may explain the low number of oligochaete species. On the other hand, Plathelminthes seem to adapt to relatively narrow ranges of factors and their species richness is highest. Because of macrofaunameiofauna interaction it is suggested that the meiofaunal assemblage will be least stable in mud and muddy sand, and most stable in exposed sand.     

Meiofaunal distribution in Hornsund fjord,Spitsbergen

The meiofaunal community structure at 32 stations in Hornsund fjord (77°N) was investigated, and results were compared with data from another Spitsbergen fjord, Kongsfjorden (79°N). Steep environmental gradients of sedimentation, organic matter content, and salinity from the inner to the outer basin of the fjord are present due to intensive glacial discharges of meltwater and ice. As the natural environmental disturbances were described for macrofauna benthic communities before, we aimed to check whether the same pattern occurs among meiofauna. A total of 12 higher meiofaunal taxa were recorded, with nematodes predominating at all stations. Non-parametric multivariate analyses demonstrated clear differences in meiofaunal abundance and composition between stations in the glacial bay and in the outer part of the fjord. Meiofaunal abundance increased with increasing distance from the source of disturbance, which in our study is tidal glaciers. Therefore, the current study demonstrates that the spatial structure of meiofauna is affected by the natural environmental disturbance, and analysis of meiofaunal assemblages can be used to assess the effect of such disturbances.     

Seasonal dynamics and spatial distribution of chydorid cladocerans in relation to chironomid larvae in the sandy littoral zone of an oligo-mesotrophic lake

The seasonal variation of the principal macro- and meiobenthic taxa in the sandy littoral zone of the oligomesotrophic Dutch Lake Maarsseveen I was studied during two years. Population peaks of the different taxa were clearly separated in time. In early spring there were chironomid density peaks of Stictochironomus sticticus, in one year followed by a Polypedilum maximum. From June–July chydorid cladocerans dominated, with a peak of Monospilus dispar followed by a peak of Rhynchotalona falcata. In autumn the chironomid Cladotanytarsus mancus became dominant, remaining so throughout the winter.To study the spatial heterogeneity of the major macro- and meiofaunal taxa, samples were taken in a grid of 2.5 × 10 m. Distributions of all but one taxon were significantly different from random, with Morisita indices varying from 1.23 (chironomids) to 2.10 (the chydorid Monospilus dispar). Wind-induced disturbance presumably had strong and species-specific effects on littoral macro- and meiobenthic taxa. Chydorid heterogeneity increased immediately following the first autumn storm of the season; this may be explained by the tendency of these organisms to remain attached to detritus particles.Temporal and spatial correlation coefficients between macro- and meiobenthic taxa were generally low, suggesting that interactions between these groups are weak, with distribution patterns that are independent of each other. Among the meiofaunal taxa, positive spatial and temporal correlation coefficients were found; apparently, the similarity in both seasonal dynamics and spatial distribution is larger among the meiofaunal taxa than between macro- and meiofaunal taxa. Also among most of the chironomid species significant positive temporal correlations were found, except for Stictochironomus sticticus and Polypedilum sp.; these species have similar life-cycles, but were during one year temporally separated by several alternating settlement peaks. In the next year, Polypedilum disappeared completely following an extremely strong Stictochironomus settlement peak.