Weekend reads

This week a hopefully eclectic collection of reads. I  also hope I posted something for everyone.

DNA metabarcoding reveals that 200-μm-size-fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes

Microeukaryotic plankton (0.2-200 μm) are critical components of aquatic ecosystems and key players in global ecological processes. High-throughput sequencing is currently revolutionizing their study on an unprecedented scale. However, it is currently unclear whether we can accurately, effectively and quantitatively depict the microeukaryotic plankton communities using traditional size-fractionated filtering combined with molecular methods. To address this, we analysed the eukaryotic plankton communities both with, and without, prefiltering with a 200 μm pore-size sieve -by using SSU rDNA-based high-throughput sequencing on 16 samples with three replicates in each sample from two subtropical reservoirs sampled from January to October in 2013. We found that ~25% reads were classified as metazoan in both size groups. The species richness, alpha and beta diversity of plankton community and relative abundance of reads in 99.2% eukaryotic OTUs showed no significant changes after prefiltering with a 200 μm pore-size sieve. We further found that both >0.2 μm and 0.2-200 μm eukaryotic plankton communities, especially the abundant plankton subcommunities, exhibited very similar, and synchronous, spatiotemporal patterns and processes associated with almost identical environmental drivers. The lack of an effect on community structure from prefiltering suggests that environmental DNA from larger metazoa is introduced into the smaller size class. Therefore, size-fractionated filtering with 200 μm is insufficient to discriminate between the eukaryotic plankton size groups in metabarcoding approaches. Our results also highlight the importance of sequencing depth, and strict quality filtering of reads, when designing studies to characterize microeukaryotic plankton communities.

eDNA metabarcoding: a promising method for anuran surveys in highly diverse tropical forests

Understanding the geographical distribution and community composition of species is crucial to monitor species persistence and define effective conservation strategies. Environmental DNA (eDNA) has emerged as a powerful noninvasive tool for species detection. However, most eDNA survey methods have been developed and applied in temperate zones. We tested the feasibility of using eDNA to survey anurans in tropical streams in the Brazilian Atlantic forest and compared the results with short-term visual and audio surveys. We detected all nine species known to inhabit our focal streams with one single visit for eDNA sampling. We found a higher proportion of sequence reads and larger number of positive PCR replicates for more common species and for those with life cycles closely associated with the streams, factors that may contribute to increased release of DNA in the water. However, less common species were also detected in eDNA samples, demonstrating the detection power of this method. Filtering larger volumes of water resulted in a higher probability of detection. Our data also show it is important to sample multiple sites along streams, particularly for detection of target species with lower population densities. For the three focal species in our study, the eDNA metabarcoding method had a greater capacity of detection per sampling event than our rapid field surveys, and thus, has the potential to circumvent some of the challenges associated with traditional approaches. Our results underscore the utility of eDNA metabarcoding as an efficient method to survey anuran species in tropical streams of the highly biodiverse Brazilian Atlantic forest.

The use of nemabiome metabarcoding to explore gastro-intestinal nematode species diversity and anthelmintic treatment effectiveness in beef calves

Next-generation deep amplicon sequencing, or metabarcoding, has revolutionized the study of microbial communities in humans, animals and the environment. However, such approaches have yet to be applied to parasitic helminth communities. We recently described the first example of such a method - nemabiome sequencing - based on deep-amplicon sequencing of internal transcribed spacer 2 (ITS-2) rDNA, and validated its ability to quantitatively assess the species composition of cattle gastro-intestinal nematode (GIN) communities. Here, we present the first application of this approach to explore GIN species diversity and the impact of anthelmintic drug treatments. First, we investigated GIN species diversity in cow-calf beef cattle herds in several different regions, using coproculture derived L3s. A screen of 50 Canadian beef herds revealed parasite species diversity to be low overall. The majority of parasite communities were comprised of just two species; Ostertagia ostertagi and Cooperia oncophora. Cooperia punctata was present at much lower levels overall, but nevertheless comprised a substantive part of the parasite community of several herds in eastern Canada. In contrast, nemabiome sequencing revealed higher GIN species diversity in beef calves sampled from central/south-eastern USA and Sao Paulo State, Brazil. In these regions C. punctata predominated in most herds with Haemonchus placei predominating in a few cases. Ostertagia ostertagi and C. oncophora were relatively minor species in these regions in contrast to the Canadian herds. We also examined the impact of routine macrocyclic lactone pour-on treatments on GIN communities in the Canadian beef herds. Low treatment effectiveness was observed in many cases, and nemabiome sequencing revealed an overall increase in the proportion of Cooperia spp. relative to O. ostertagi post-treatment. This work demonstrates the power of nemabiome metabarcoding to provide a detailed picture of GIN parasite community structure in large sample sets and illustrates its potential use in research, diagnostics and surveillance.

Minimizing polymerase biases in metabarcoding

DNA metabarcoding is an increasingly popular method to characterize and quantify biodiversity in environmental samples. Metabarcoding approaches simultaneously amplify a short, variable genomic region, or "barcode," from a broad taxonomic group via the polymerase chain reaction (PCR), using universal primers that anneal to flanking conserved regions. Results of these experiments are reported as occurrence data, which provide a list of taxa amplified from the sample, or relative abundance data, which measure the relative contribution of each taxon to the overall composition of amplified product. The accuracy of both occurrence and relative abundance estimates can be affected by a variety of biological and technical biases. For example, taxa with larger biomass may be better represented in environmental samples than those with smaller biomass. Here, we explore how polymerase choice, a potential source of technical bias, might influence results in metabarcoding experiments. We compared potential biases of six commercially available polymerases using a combination of mixtures of amplifiable synthetic sequences and real sedimentary DNA extracts. We find that polymerase choice can affect both occurrence and relative abundance estimates and that the main source of this bias appears to be polymerase preference for sequences with specific GC contents. We further recommend an experimental approach for metabarcoding based on results of our synthetic experiments.

Molecular gut content analysis of different spider body parts

Molecular gut-content analysis has revolutionized the study of food webs and feeding interactions, allowing the detection of prey DNA within the gut of many organisms. However, successful prey detection is a challenging procedure in which many factors affect every step, starting from the DNA extraction process. Spiders are liquid feeders with branched gut diverticula extending into their legs and throughout the prosoma, thus digestion takes places in different parts of the body and simple gut dissection is not possible. In this study, we investigated differences in prey detectability in DNA extracts from different parts of the spider´s body: legs, prosoma and opisthosoma, using prey-specific PCR and metabarcoding approaches. We performed feeding trials with the woodlouse hunter spider Dysdera verneaui Simon, 1883 (Dysderidae) to estimate the time at which prey DNA is detectable within the predator after feeding. Although we found that all parts of the spider body are suitable for gut-content analysis when using prey-specific PCR approach, results based on metabarcoding suggested the opisthosoma is optimal for detection of predation in spiders because it contained the highest concentration of prey DNA for longer post feeding periods. Other spiders may show different results compared to D. verneaui, but given similarities in the physiology and digestion in different families, it is reasonable to assume this to be common across species and this approach having broad utility across spiders.

Consistent patterns of high alpha and low beta diversity in tropical parasitic and free-living protists

Tropical animals and plants are known to have high alpha diversity within forests, but low beta diversity between forests. By contrast, it is unknown if microbes inhabiting the same ecosystems exhibit similar biogeographic patterns. To evaluate the biogeographies of tropical protists, we used metabarcoding data of species sampled in the soils of three lowland Neotropical rainforests. Taxa-area and distance-decay relationships for three of the dominant protist taxa and their subtaxa were estimated at both the OTU- and phylogenetic-levels, with presence-absence and abundance based measures. These estimates were compared to null models. High local alpha and low regional beta diversity patterns were consistently found for both the parasitic Apicomplexa and the largely free-living Cercozoa and Ciliophora. Similar to animals and plants, the protists showed spatial structures between forests at the OTU- and phylogenetic-levels, and only at the phylogenetic level within forests. These results suggest that the biogeographies of macro- and micro-organismal eukaryotes in lowland Neotropical rainforests are partially structured by the same general processes. However, and unlike the animals and plants, the protist OTUs did not exhibit spatial structures within forests, which hinders our ability to estimate local and regional diversity of protists in tropical forests.

DNA metabarcoding diet analysis reveals dynamic feeding behaviour and biological control potential of carabid farmland communities (preprint)

Maximizing the delivery of key ecosystem services such as biological control through the management of natural enemy communities is one of the major challenges for modern agriculture. The main obstacle lies in our yet limited capacity of identifying the factors that drive the dynamics of trophic interactions within multi-species assemblages. Invertebrate generalist predators like carabid beetles are known for their dynamic feeding behaviour. Yet, at what extent different carabid species contribute to the regulation of animal and plant pests within agroecosystems is currently unknown. Here, we developed a DNA metabarcoding approach for characterizing the full diet spectrum of a community of fourteen very common carabid species inhabiting an intensively managed Western-European agroecosystem. We then investigated how diet and biological control potential within the carabid community varies with the sampling field location and the crop type (wheat vs oilseed rape). DNA metabarcoding diet analysis allowed to detect a wide variety of animal and plant taxa from carabid gut contents thus confirming their generalist feeding behaviour. The most common prey categories detected were arachnids, insects, earthworms and several plant families potentially including many weed species. Our results also show that the field location and the crop type are much stronger determinants then the species regarding carabid dietary choice: significantly more trophic links involving dipteran prey were observed in wheat, whereas more collembolan and plant prey was consumed in oilseed rape by the same carabid community. We speculate that structural differences in the habitats provided by these two crop types drive differences in resource availability cascading up the trophic chain, and we assume that specific carabid taxa could hardly be used to infer levels of ecosystem services (biological control) or disservices (e.g. intraguild predation). However, as this is the first study to report the use of DNA metabarcoding diet analysis in predatory carabid beetles we urge caution over the interpretation of our results. For instance, overall detection rates were rather low (31% of the individuals analysed tested positive for at least one prey category) most likely due to the overwhelming amplification of the carabid host DNA. Therefore, we acknowledge that more studies are required in order to confirm our observations and conclude with few recommendations for further improvements of the community-level DNA metabarcoding analysis of carabid diet.

Brazil - new legislation with dire consequences

The Brazilian Federal Government has put their so called New Law on Biodiversity into effect and it seems it is a huge step backwards with severe implications for the Brazilian colleagues and biodiversity research overall.

The key section of this new legislation that was met with some strong but healthy criticism is the fact that literally every bit of research activity done on Brazilian biodiversity must now be registered in the National System of Genetic Resource Management and Associated Traditional Knowledge (SisGen). Any dissemination of research results that were not registered in SisGen, even in conference talks, or a shipment made without prior registration, will represent infractions subject to hefty fines (up to US$ 3,000,000 for legal entities). In order to do so each institution needs a legal representative who will then be in sole control of registering researchers as applicants to SisGen.

A group of Brazilian colleagues took this to Science and made clear, that if not repealed or substantially overhauled, this Byzantine labyrinth of unnecessary demands and threats will decimate scientific research on Brazilian biodiversity by requiring scientists to divert an inordinate amount of already limited resources from research to the time-consuming process of  registering every specimen, DNA sequence, photograph, and any other observation of Brazilian biodiversity before publication, presentation at scientific meetings, or dissemination to media outlets.

I am fairly certain that biodiversity research in Brazil will come to a grinding halt especially when international collaborations are involved. Even worse the law is working retroactively going back as far as 2000. 

Proponents of the law react to the criticism in a rather condescending manner:

Despite the opposition of some academics about government control over research involving Brazilian biodiversity, due to the resulting bureaucratization, it is important to clarify that this control was foreseen in the Federal Constitution of 1988, as well as in the Convention on Biological Diversity (CBD) and the Nagoya Protocol (supplementary agreement to the CBD), which aim to safeguard the conservation of biological diversity, the sustainable use of its components, and the rights of holders of associated traditional knowledge, as well as the fair and equitable sharing of the benefits arising from the utilization of genetic resources and associated traditional knowledge.

I am not so sure if this new law is in line with the goals of the CBD and their intentions for Access and Benefit Sharing. Brazil shouldn't be surprised if there is pushback from other member states in a similar situation that have far more progressive legislation in place. What is infuriating though is the fact that commercial activities involving Brazilian biodiversity, such as the export of ornamental fishes, tropical plants, grains, and other marketable products, remain unaffected by the law. That doesn't sound like safeguarding national biodiversity to me .

From the inbox: Help with mystery fungus of the Arctic

Found this in my inbox yesterday and thought it couldn't hurt to spread the word further. Tomas Roslin wrote:

Kadri Pärtel from the Univeristy of Tartu is asking for help from good ecologists travelling in the Arctic this summer.

Kadri is keen to obtain samples and pictures of some ascomycetes growing gregariously on Diapensia lapponica dead leaves in summer (June, July, August). These fungi have semitranslucent-beige turbinate cup-like fruitbodies, 100-250 um in diam. Based on material collected in 1970-1980s the preliminary description of this new taxon was made by Estonian mycologist Ain Raitviir, but Kadri would now need additional information - a recent specimen for DNA barcoding and phylogeny as well as for making illustrations to describe this species with yet unclear generic affiliation.

Here a photo of something similar (a different fungus though) to what you should look for. In brief: translucent, really tiny <0.5 mm cups on dead Diapensia leaves. An in situ photo would be very nice.

Please keep an eye out for these fungi! Greenland may be a promising spot.

Weekend reads

After some crazily busy weeks a quick weekend read blog post. Some really good papers have appeared in the last couple of weeks. Hard to make a selection.

A MinION-based pipeline for fast and cost-effective DNA barcoding

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory, is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrate that one flowcell can generate barcodes for ~500 specimens despite the high base-call error rates of MinION™ reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that are corrected based on conserved amino-acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (1) 511 specimens of a mixed Diptera sample, (2) 575 specimens of ants, and (3) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N=471). Overall, the MinION barcodes have an accuracy of 99.3%-100% with the number of ambiguous bases after correction ranging from <0.01-1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hours of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1000 barcodes can be generated in one flowcell and that the cost per barcode can be <USD 2. 

Stable baselines of temporal turnover underlie high beta diversity in tropical arthropod communities

While the high species diversity of tropical arthropod communities has often been linked to marked spatial heterogeneity, their temporal dynamics have received little attention. This study addresses this gap by examining spatio-temporal variation in the arthropod communities of a tropical montane forest in Honduras. By employing DNA barcode analysis and Malaise trap sampling across four years and five sites, 51,596 specimens were assigned to 8,193 presumptive species. High beta diversity was linked more strongly to elevation than geographic distance, decreasing by 12% when only the dominant species were considered. When sampling effort was increased by deploying more traps at a site, beta diversity only decreased by 2%, but extending sampling across years decreased beta diversity by 27%. Species inconsistently detected among years, likely transients from other settings, drove the low similarity in species composition among traps only a few metres apart. The dominant, temporally persistent species substantially influenced the cyclic pattern of change in community composition among years. This pattern likely results from divergence-convergence dynamics, suggesting a stable baseline of temporal turnover in each community. The overall results establish that large sample sizes are necessary to reveal species richness, but are not essential for quantifying beta diversity. This study further highlights the need for standardized methods of sampling and species identification to generate the comparative data required to evaluate biodiversity change in space and time.

Natural history bycatch: a pipeline for identifying metagenomic sequences in RADseq data

BACKGROUND:

Reduced representation genomic datasets are increasingly becoming available from a variety of organisms. These datasets do not target specific genes, and so may contain sequences from parasites and other organisms present in the target tissue sample. In this paper, we demonstrate that (1) RADseq datasets can be used for exploratory analysis of tissue-specific metagenomes, and (2) tissue collections house complete metagenomic communities, which can be investigated and quantified by a variety of techniques.

METHODS:

We present an exploratory method for mining metagenomic "bycatch" sequences from a range of host tissue types. We use a combination of the pyRAD assembly pipeline, NCBI's blastn software, and custom R scripts to isolate metagenomic sequences from RADseq type datasets.

RESULTS:

When we focus on sequences that align with existing references in NCBI's GenBank, we find that between three and five percent of identifiable double-digest restriction site associated DNA (ddRAD) sequences from host tissue samples are from phyla to contain known blood parasites. In addition to tissue samples, we examine ddRAD sequences from metagenomic DNA extracted snake and lizard hind-gut samples. We find that the sequences recovered from these samples match with expected bacterial and eukaryotic gut microbiome phyla.

DISCUSSION:

Our results suggest that (1) museum tissue banks originally collected for host DNA archiving are also preserving valuable parasite and microbiome communities, (2) that publicly available RADseq datasets may include metagenomic sequences that could be explored, and (3) that restriction site approaches are a useful exploratory technique to identify microbiome lineages that could be missed by primer-based approaches.

Remarkable fly (Diptera) diversity in a patch of Costa Rican cloud forest: Why inventory is a vital science.

Study of all flies (Diptera) collected for one year from a four-hectare (150 x 266 meter) patch of cloud forest at 1,600 meters above sea level at Zurquí de Moravia, San José Province, Costa Rica (hereafter referred to as Zurquí), revealed an astounding 4,332 species. This amounts to more than half the number of named species of flies for all of Central America. Specimens were collected with two Malaise traps running continuously and with a wide array of supplementary collecting methods for three days of each month. All morphospecies from all 73 families recorded were fully curated by technicians before submission to an international team of 59 taxonomic experts for identification.        Overall, a Malaise trap on the forest edge captured 1,988 species or 51% of all collected dipteran taxa (other than of Phoridae, subsampled only from this and one other Malaise trap). A Malaise trap in the forest sampled 906 species. Of other sampling methods, the combination of four other Malaise traps and an intercept trap, aerial/hand collecting, 10 emergence traps, and four CDC light traps added the greatest number of species to our inventory. This complement of sampling methods was an effective combination for retrieving substantial numbers of species of Diptera. Comparison of select sampling methods (considering 3,487 species of non-phorid Diptera) provided further details regarding how many species were sampled by various methods. Comparison of species numbers from each of two permanent Malaise traps from Zurquí with those of single Malaise traps at each of Tapantí and Las Alturas, 40 and 180 km distant from Zurquí respectively, suggested significant species turnover. Comparison of the greater number of species collected in all traps from Zurquí did not markedly change the degree of similarity between the three sites, although the actual number of species shared did increase. Comparisons of the total number of named and unnamed species of Diptera from four hectares at Zurquí is equivalent to 51% of all flies named from Central America, greater than all the named fly fauna of Colombia, equivalent to 14% of named Neotropical species and equal to about 2.7% of all named Diptera worldwide. Clearly the number of species of Diptera in tropical regions has been severely underestimated and the actual number may surpass the number of species of Coleoptera. Various published extrapolations from limited data to estimate total numbers of species of larger taxonomic categories (e.g., Hexapoda, Arthropoda, Eukaryota, etc.) are highly questionable, and certainly will remain uncertain until we have more exhaustive surveys of all and diverse taxa (like Diptera) from multiple tropical sites. Morphological characterization of species in inventories provides identifications placed in the context of taxonomy, phylogeny, form, and ecology. DNA barcoding species is a valuable tool to estimate species numbers but used alone fails to provide a broader context for the species identified.

Using metabarcoding to reveal and quantify plant-pollinator interactions

Given the ongoing decline of both pollinators and plants, it is crucial to implement effective methods to describe complex pollination networks across time and space in a comprehensive and high-throughput way. Here we tested if metabarcoding may circumvent the limits of conventional methodologies in detecting and quantifying plant-pollinator interactions. Metabarcoding experiments on pollen DNA mixtures described a positive relationship between the amounts of DNA from focal species and the number of trnL and ITS1 sequences yielded. The study of pollen loads of insects captured in plant communities revealed that as compared to the observation of visits, metabarcoding revealed 2.5 times more plant species involved in plant-pollinator interactions. We further observed a tight positive relationship between the pollen-carrying capacities of insect taxa and the number of trnL and ITS1 sequences. The number of visits received per plant species also positively correlated to the number of their ITS1 and trnL sequences in insect pollen loads. By revealing interactions hard to observe otherwise, metabarcoding significantly enlarges the spatiotemporal observation window of pollination interactions. By providing new qualitative and quantitative information, metabarcoding holds great promise for investigating diverse facets of interactions and will provide a new perception of pollination networks as a whole.

Inferring neutral biodiversity parameters using environmental DNA data sets

The DNA present in the environment is a unique and increasingly exploited source of information for conducting fast and standardized biodiversity assessments for any type of organisms. The datasets resulting from these surveys are however rarely compared to the quantitative predictions of biodiversity models. In this study, we simulate neutral taxa-abundance datasets, and artificially noise them by simulating noise terms typical of DNA-based biodiversity surveys. The resulting noised taxa abundances are used to assess whether the two parameters of Hubbell's neutral theory of biodiversity can still be estimated. We find that parameters can be inferred provided that PCR noise on taxa abundances does not exceed a certain threshold. However, inference is seriously biased by the presence of artifactual taxa. The uneven contribution of organisms to environmental DNA owing to size differences and barcode copy number variability does not impede neutral parameter inference, provided that the number of sequence reads used for inference is smaller than the number of effectively sampled individuals. Hence, estimating neutral parameters from DNA-based taxa abundance patterns is possible but requires some caution. In studies that include empirical noise assessments, our comprehensive simulation benchmark provides objective criteria to evaluate the robustness of neutral parameter inference.

Molecular evolution of a widely-adopted taxonomic marker (COI) across the animal tree of life.

DNA barcodes are widely used for identification and discovery of species. While such use draws on information at the DNA level, the current amassment of ca. 4.7 million COI barcodes also offers a unique resource for exploring functional constraints on DNA evolution. Here, we explore amino acid variation in a crosscut of the entire animal kingdom. Patterns of DNA variation were linked to functional constraints at the level of the amino acid sequence in functionally important parts of the enzyme. Six amino acid sites show variation with possible effects on enzyme function. Overall, patterns of amino acid variation suggest convergent or parallel evolution at the protein level connected to the transition into a parasitic life style. Denser sampling of two diverse insect taxa revealed that the beetles (Coleoptera) show more amino acid variation than the butterflies and moths (Lepidoptera), indicating fundamental difference in patterns of molecular evolution in COI. Several amino acid sites were found to be under notably strong purifying selection in Lepidoptera as compared to Coleoptera. Overall, these findings demonstrate the utility of the global DNA barcode library to extend far beyond identification and taxonomy, and will hopefully be followed by a multitude of work.

Construction, validation and application of nocturnal pollen transport networks in an agro-ecosystem: a comparison using microscopy and DNA metabarcoding (preprint)

Moths are globally relevant as pollinators but nocturnal pollination remains poorly understood. Plant-pollinator interaction networks are traditionally constructed using either flower-visitor observations or pollen-transport detection using microscopy. Recent studies have shown the potential of DNA metabarcoding for detecting and identifying pollen-transport interactions. However, no study has directly compared the realised observations of pollen-transport networks between DNA metabarcoding and conventional light microscopy. Using matched samples of nocturnal moths, we construct pollen-transport networks using two methods: light microscopy and DNA metabarcoding. Focussing on the feeding mouthparts of moths, we develop and provide reproducible methods for merging DNA metabarcoding and ecological network analysis to better understand species-interactions. DNA metabarcoding detected pollen on more individual moths, and detected multiple pollen types on more individuals than microscopy, but the average number of pollen types per individual was unchanged. However, after aggregating individuals of each species, metabarcoding detected more interactions per moth species. Pollen-transport network metrics differed between methods, because of variation in the ability of each to detect multiple pollen types per moth and to separate morphologically-similar or related pollen. We detected unexpected but plausible moth-plant interactions with metabarcoding, revealing new detail about nocturnal pollination systems. The nocturnal pollination networks observed using metabarcoding and microscopy were similar, yet distinct, with implications for network ecologists. Comparisons between networks constructed using metabarcoding and traditional methods should therefore be treated with caution. Nevertheless, the potential applications of metabarcoding for studying plant-pollinator interaction networks are encouraging, especially when investigating understudied pollinators such as moths.

Biofilm biodiversity in French and Swiss show caves using the metabarcoding approach: First data

In recent decades, show caves have begun to suffer from microorganism proliferation due to artificial lighting installations for touristic activity. In addition to the aesthetic problem, light encourages microorganisms that are responsible for physical and chemical degradation of limestone walls, speleothems and prehistoric paintings of cultural value. Microorganisms have previously been described by microscopy or culture-dependent methods, but data provided by new generation sequencing are rare. The authors identified, for the first time, microorganisms proliferating in one Swiss and in four French show caves using three different primers. The results showed that both photosynthetic and non-photosynthetic bacteria were the dominant taxa present in biofilms. Microalgae were heavily represented by the Trebouxiophyceae, Eustigmatophyceae and Chlorophyceae groups. Twelve diatoms were also recorded, with dominance of Syntrichia sp. (96.1%). Fungi were predominantly represented by Ascomycota, Zygomycota and Basidiomycota, fully half of the sampled biofilms where Fungi were detected. Comparing microbial communities from bleach-treated caves to those in untreated caves showed no significant difference except for a low-level change in the abundance of certain taxa. These findings provided by Illumina sequencing reveal a complex community structure in the 5 caves based on the assembly of bacteria, cyanobacteria, algae, diatoms, fungi and mosses.

First circular for iBOL2019

Dear friends and colleagues,

We invite you to visit and bookmark the 8th International Barcode of Life Conference webpage. Please save the dates June 17-20, 2019 in your calendars.

The scientific and social programs are under development, but we are confident that both will be memorable!

Please distribute this invitation broadly in your network and stay tuned for updates.

Heineken Prize for Paul Hebert

The Heineken Prizes are the most prestigious international science prizes of the Netherlands. They are awarded every other year. The laureates are selected by juries assembled by the Royal Netherlands Academy of Arts and Sciences and made up of leading Dutch and foreign scientists and scholars. The Heineken Prizes are named after Dr Henry P. Heineken (1886-1971); Dr Alfred H. Heineken (1923-2002) and Charlene de Carvalho-Heineken (1954), chairman of the Dr H.P. Heineken Foundation and the Alfred Heineken Fondsen Foundation, which fund the prizes.

The Academy has awarded the Heineken Prizes this year to biomedical scientist Peter Carmeliet (University of Leuven), cognitive scientist Nancy Kanwisher (MIT), historian John R. McNeill (Georgetown University), biophysicist Xiaowei Zhuang (Harvard University) and - here it comes - biologist Paul Hebert (University of Guelph).

A great and well deserved honour for Paul, the father of DNA barcoding (I know he hates this title but I couldn't resist teasing him).

Weekend reads

Lots of work and distractions keep me from blogging these days. Hope to get back to old routine in the coming weeks. Meanwhile, some more papers to read:

Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR-free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (3 different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome-skimming) respectively. We found that even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read-biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for 8 out of 13 (amplicons B1FR-450bp, FF130R-130bp) or 4 out of 13 (amplicon FFFR, 658bp) species. Combining the results of all three COI amplicons (multi-amplicon approach) improved the read-biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.

Estimating intraspecific genetic diversity from community DNA metabarcoding data

BACKGROUND: DNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high-throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTUs), losing information on intraspecific diversity in the process. While Cytochrome c oxidase subunit I (COI) haplotype information is limited in resolving intraspecific diversity it is nevertheless often useful e.g. in a phylogeographic context, helping to formulate hypotheses on taxon distribution and dispersal.

METHODS: This study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotype information from freshwater macroinvertebrate metabarcoding datasets. This novel approach was added to the R package "JAMP" and can be applied to COI amplicon datasets. We tested our haplotyping method by sequencing (i) a single-species mock community composed of 31 individuals with 15 different haplotypes spanning three orders of magnitude in biomass and (ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates.

RESULTS: We detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177-200 OTUs, each containing an average of 2.40-3.30 haplotypes per OTU. The derived intraspecific diversity data showed population structures that were consistent between replicates and similar between primer pairs but resolution depended on the primer length. A closer look at abundant taxa in the dataset revealed various population genetic patterns, e.g. the stonefly Taeniopteryx nebulosa and the caddisfly Hydropsyche pellucidula showed a distinct north-south cline with respect to haplotype distribution, while the beetle Oulimnius tuberculatus and the isopod Asellus aquaticus displayed no clear population pattern but differed in genetic diversity.

DISCUSSION: We developed a strategy to infer intraspecific genetic diversity from bulk invertebrate metabarcoding data. It needs to be stressed that at this point this metabarcoding-informed haplotyping is not capable of capturing the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding datasets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about species diversity but also underlying genetic diversity.

Phylogeographic structure in three North American tent caterpillar species (Lepidoptera: Lasiocampidae): Malacosoma americana, M. californica, and M. disstria.

While phylogeographic structure has been examined in many North American vertebrate species, insects have received much less attention despite their central ecological roles. The moth genus Malacosoma (Hübner, 1820), is an important group of forestry pests responsible for large-scale defoliation across much of the Nearctic and Palearctic. The present study uses sequence variation in the mitochondrial cytochrome c oxidase 1 (COI) gene to examine the population genetic structure of the three widespread Malacosoma species (M. americana, M. californica, and M. disstria). Populations of all three species showed highest diversity in the south, suggesting that modern populations derived from southern refugia with loss of variation as these lineages dispersed northwards. However, despite similar life histories and dispersal abilities, the extent of regional variation varied among the taxa. M. americana, a species restricted to eastern North America, showed much less genetic structure than the western M. californica or the widespread M. disstria. The regional differentiation in the latter reflects the likely derivation of modern lineages from several refugia, as well as taxonomic uncertainty in M. californica. In these respects, the three species of Malacosoma share phylogeographic patterns similar to those detected in vertebrates which are characterised by greater phylogeographic breaks in the western half of the continent and limited structure in the east.

DNA metabarcoding and microscopic analyses of sea turtles biofilms: Complementary to understand turtle behavior

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building

BACKGROUND: Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field.

FINDINGS: We demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding.

CONCLUSIONS: Overall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive

Assessment of ecological status for the European Water Framework Directive (WFD) is based on "Biological Quality Elements" (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identification of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches. We rate the applicability of DNA-based identification for the individual BQEs and water categories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identification (knowledge about uncertainty), comparability with conventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environmental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electrofishing. Furthermore, there are attempts to replace absolute by relative abundance in metric calculations. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss general implications of implementing DNA-based identification into standard ecological assessment, in particular considering any adaptations to the WFD that may be required to facilitate the transition to molecular data.

High-throughput terrestrial biodiversity assessments: mitochondrial metabarcoding, metagenomics or metatranscriptomics?

Consensus on the optimal high-throughput sequencing (HTS) approach to examine biodiversity in mixed terrestrial arthropod samples has not been reached. Metatranscriptomics could increase the proportion of taxonomically informative mitochondrial reads in HTS outputs but has not been investigated for terrestrial arthropod samples. We compared the efficiency of 16S rRNA metabarcoding, metagenomics and metatranscriptomics for detecting species in a mixed terrestrial arthropod sample (pooled DNA/RNA from 38 taxa). 16S rRNA metabarcoding and nuclear rRNA-depleted metatranscriptomics had the highest detection rate with 97% of input species detected. Based on cytochrome c oxidase I, metagenomics had the highest detection rate with 82% of input species detected, but metatranscriptomics produced a larger proportion of reads matching (Sanger) reference sequences. Metatranscriptomics with nuclear rRNA depletion may offer advantages over metabarcoding through reducing the number of spurious operational taxonomic units while retaining high detection rates, and offers natural enrichment of mitochondrial sequences which may enable increased species detection rates compared with metagenomics.

Environmental DNA filtration techniques affect recovered biodiversity

Freshwater metazoan biodiversity assessment using environmental DNA (eDNA) captured on filters offers new opportunities for water quality management. Filtering of water in the field is a logistical advantage compared to transport of water to the nearest lab, and thus, appropriate filter preservation becomes crucial for maximum DNA recovery. Here, the effect of four different filter preservation strategies, two filter types, and pre-filtration were evaluated by measuring metazoan diversity and community composition, using eDNA collected from a river and a lake ecosystem. The filters were preserved cold on ice, in ethanol, in lysis buffer and dry in silica gel. Our results show that filters preserved either dry or in lysis buffer give the most consistent community composition. In addition, mixed cellulose ester filters yield more consistent community composition than polyethersulfone filters, while the effect of pre-filtration remained ambiguous. Our study facilitates development of guidelines for aquatic community-level eDNA biomonitoring, and we advocate filtering in the field, using mixed cellulose ester filters and preserving the filters either dry or in lysis buffer.

Counting with DNA in metabarcoding studies: how should we convert sequence reads to dietary data? (Preprint)

Advances in DNA sequencing technology have revolutionised the field of molecular analysis of trophic interactions and it is now possible to recover counts of food DNA barcode sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of the overall diet of a consumer should we work strictly with datasets summarising the frequency of occurrence of different food taxa, or is it possible to use the relative number of sequences? Both approaches are applied in the dietary metabarcoding literature, but occurrence data is often promoted as a more conservative and reliable option due to taxa-specific biases in recovery of sequences. Here, we point out that diet summaries based on occurrence data overestimate the importance of food consumed in small quantities (potentially including low-level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that even with recovery biases incorporated, using relative read abundance (RRA) information can provide a more accurate view of population-level diet in many scenarios. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue to addressing methodological challenges, and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research.

Plant DNA metabarcoding of lake sediments: How does it represent the contemporary vegetation

Metabarcoding of lake sediments have been shown to reveal current and past biodiversity, but little is known about the degree to which taxa growing in the vegetation are represented in environmental DNA (eDNA) records. We analysed composition of lake and catchment vegetation and vascular plant eDNA at 11 lakes in northern Norway. Out of 489 records of taxa growing within 2 m from the lake shore, 17-49% (mean 31%) of the identifiable taxa recorded were detected with eDNA. Of the 217 eDNA records of 47 plant taxa in the 11 lakes, 73% and 12% matched taxa recorded in vegetation surveys within 2 m and up to about 50 m away from the lakeshore, respectively, whereas 16% were not recorded in the vegetation surveys of the same lake. The latter include taxa likely overlooked in the vegetation surveys or growing outside the survey area. The percentages detected were 61, 47, 25, and 15 for dominant, common, scattered, and rare taxa, respectively. Similar numbers for aquatic plants were 88, 88, 33 and 62%, respectively. Detection rate and taxonomic resolution varied among plant families and functional groups with good detection of e.g. Ericaceae, Roseaceae, deciduous trees, ferns, club mosses and aquatics. The representation of terrestrial taxa in eDNA depends on both their distance from the sampling site and their abundance and is sufficient for recording vegetation types. For aquatic vegetation, eDNA may be comparable with, or even superior to, in-lake vegetation surveys and may therefore be used as an tool for biomonitoring. For reconstruction of terrestrial vegetation, technical improvements and more intensive sampling is needed to detect a higher proportion of rare taxa although DNA of some taxa may never reach the lake sediments due to taphonomical constrains. Nevertheless, eDNA performs similar to conventional methods of pollen and macrofossil analyses and may therefore be an important tool for reconstruction of past vegetation.