New Issue of the Bulletin

A lot of things happened this year and some of those clearly had an impact on our ability to supply the community with their fair share of barcode bulletins. After some restructuring we are back on track and here is this year's December issue:

Just click on the image to download and enjoy reading.

only 7 days to go

Only seven days left until the 7th International Barcode of Life conference starts in Kruger National Park. The conference program is currently finalized. If you are curious you can get a sense here.

The abstract volume published by Genome has also appeared today. Seems we are all set to go.

Safe travels to all delegates!

We will see each other here in a week.

Two Postdocs at Naturalis Biodiversity Center

Naturalis Biodiversity Center is searching for

Two Postdocs to study spatial and temporal variability of marine biodiversity using an eDNA or metabarcoding approach

In collaboration with the Institute of Environmental Sciences at Leiden University (CML), Naturalis

develops the Netherlands Center for Genetic Biodiversity Assessments, combining world leading expertise on taxonomy, ecology and genetics. Using our extended DNA reference collections, our high-tech lab facilities and our bioinformatics pipelines, we continuously improve and apply genetic biodiversity assessments, making them more rapid, accurate, reliable and cost effective. Applications are in freshwater quality, marine benthic surveys, soil fertility diagnosis, air pollen distribution or invasive species surveillance. Genetic assessments are already widely used for impact analyses.

To strengthen our marine activities on DNA biomonitoring, Naturalis is searching for two postdocs within the research team Marine Biodiversity. The postdocs will execute their own research projects and thereby contribute to the optimization of molecular techniques and protocols for genetic identification and detection of marine species and communities. They will also develop new proposals for additional funding.

Qualifications:
● a PhD in a for this position relevant discipline;
● a strong background and track-record in metabarcoding or eDNA techniques;
● experience with the identification and taxonomy of one or more marine taxa (no limitation to
particular groups – could be zoological, botanical, algological, etc.), and the willingness to work on others;
● experience in the North Sea or NW European waters is preferred but not required;
● experience in grant writing and demonstrated ability to acquire external funding;
● at least three years of postdoc experience.

What we offer

A contract (36 hours per week) for a period of one year, to be extended with one year after successful

first year evaluation. A salary of circa € 3.000- 4.000 gross per month, circa €40.000- 53.300 gross per year, depending on experience. As an employee of Naturalis you will work under the Collective Bargaining Agreement of Dutch Museums. Naturalis Biodiversity Center promotes gender equality and wants to enhance the diversity of staff members. Feel free to contact Dr. Willem Renema or Berry van der Hoorn with questions about the position.

Procedure

Applicants are invited to submit their application, including a cover letter, CV (should include: (1) complete publication list with IFs -when relevant-, number of citations, and your H-Index; (2) grants obtained; (3) teaching experience; (4) invited talks; (5) other relevant information), and research statement (max 2.5 A4, should include 1) outline of the intended project to be conducted during two year appointment, 2) link and contribution to the Naturalis goals described, 3) time frame and 4) requirements/necessities needed to successfully complete project) and the names and e-mail addresses of at least two persons that can be contacted for reference before October 27th using the application form .

Metabarcoding and Metagenomics Journal is out

As announced before the new journal Metabarcoding and Metagenomics has been created by Pensoft and now it is officially running with the first few articles published. Here a part of the official press release:

A new innovative open-access academic journal Metabarcoding and Metagenomics (MBMG) is launched to welcome novel papers from both basic and applied aspects.

Focusing on genetic approaches to study biodiversity across all ecosystems, MBMG covers a considerably large scope of research including environmental, microbial and applied metabarcoding and metagenomics (especially DNA-based bioassessment and -monitoring, quarantine, nature conservation, species invasions, eDNA surveillance), as well as associated topics, such as molecular ecology, DNA-based species delimitation and identification, and other emerging related fields. Submissions of bioinformatic approaches to MBMG (algorithms, software) are also encouraged.

Featuring novel article formats and data publishing workflows, MBMG is to reflect the rapid growth in the use of metabarcoding and metagenomics in life and environmental sciences.

For the full release please read here.

I am serving as deputy Editor-in-chief for the journal and I am really looking forward to the deluge of publications to come.

Weekend reads

More to read including some from the rather large backlog. Have a great weekend with some good reads.

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case.

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5' terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5' region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding

Environmental bulk samples often contain many different taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high-throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size (as a proxy for biomass) and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here, we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate bulk samples, collected from a low-mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5 × 5, 5 × 10, and up to 10 × 20 mm). Tissue powder from each size category was extracted individually and pooled based on tissue weight to simulate samples that were not sorted by biomass ("Unsorted"). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass ("Sorted"). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately fivefold when sorting the samples into three size classes and pooling by specimen abundance. Even coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high-throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass or size is a simple yet efficient method to reduce current sequencing costs.

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives

Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and phylogenetic information for species discrimination and taxonomic assignment. Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples. Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer-template mismatches bias the taxonomic profile when using regular and highly degenerate primers. The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.

Benefits and Limitations of DNA Barcoding and Metabarcoding in Herbal Product Authentication.

INTRODUCTION:

Herbal medicines play an important role globally in the health care sector and in industrialised countries they are often considered as an alternative to mono-substance medicines. Current quality and authentication assessment methods rely mainly on morphology and analytical phytochemistry-based methods detailed in pharmacopoeias. Herbal products however are often highly processed with numerous ingredients, and even if these analytical methods are accurate for quality control of specific lead or marker compounds, they are of limited suitability for the authentication of biological ingredients.

OBJECTIVE:

To review the benefits and limitations of DNA barcoding and metabarcoding in complementing current herbal product authentication.

METHOD:

Recent literature relating to DNA based authentication of medicinal plants, herbal medicines and products are summarised to provide a basic understanding of how DNA barcoding and metabarcoding can be applied to this field.

RESULTS:

Different methods of quality control and authentication have varying resolution and usefulness along the value chain of these products. DNA barcoding can be used for authenticating products based on single herbal ingredients and DNA metabarcoding for assessment of species diversity in processed products, and both methods should be used in combination with appropriate hyphenated chemical methods for quality control.

CONCLUSIONS:

DNA barcoding and metabarcoding have potential in the context of quality control of both well and poorly regulated supply systems. Standardisation of protocols for DNA barcoding and DNA sequence-based identification are necessary before DNA-based biological methods can be implemented as routine analytical approaches and approved by the competent authorities for use in regulated procedures. 

Environment and host identity structure communities of green algal symbionts in lichens

An understanding of how biotic interactions shape species' distributions is central to predicting host-symbiont responses under climate change. Switches to locally adapted algae have been proposed to be an adaptive strategy of lichen-forming fungi to cope with environmental change. However, it is unclear how lichen photobionts respond to environmental gradients, and whether they play a role in determining the fungal host's upper and lower elevational limits. Deep-coverage Illumina DNA metabarcoding was used to track changes in the community composition of Trebouxia algae associated with two phylogenetically closely related, but ecologically divergent fungal hosts along a steep altitudinal gradient in the Mediterranean region. We detected the presence of multiple Trebouxia species in the majority of thalli. Both altitude and host genetic identity were strong predictors of photobiont community assembly in these two species. The predominantly clonally dispersing fungus showed stronger altitudinal structuring of photobiont communities than the sexually reproducing host. Elevation ranges of the host were not limited by the lack of compatible photobionts. Our study sheds light on the processes guiding the formation and distribution of specific fungal-algal combinations in the lichen symbiosis. The effect of environmental filtering acting on both symbiotic partners appears to shape the distribution of lichens.

Weekend reads

Back after a longer hiatus with more reads for you. Too much work and a little bit of vacation in between didn't allow for much posting. Let's if some reshuffling of things work better. Well, enough about me, back to other's papers (although the first is mine ;-) ):

DNA analysis of traded shark fins and mobulid gill plates reveals a high proportion of species of conservation concern

Continuously increasing demand for plant and animal products causes unsustainable depletion of biological resources. It is estimated that one-quarter of sharks and rays are threatened worldwide and although the global fin trade is widely recognized as a major driver, demand for meat, liver oil, and gill plates also represents a significant threat. This study used DNA barcoding and 16 S rRNA sequencing as a method to identify shark and ray species from dried fins and gill plates, obtained in Canada, China, and Sri Lanka. 129 fins and gill plates were analysed and searches on BOLD produced matches to 20 species of sharks and five species of rays or – in two cases – to a species pair. Twelve of the species found are listed or have been approved for listing in 2017 in the appendices of the Convention on International Trade in Endangered Species of Fauna and Flora (CITES), including the whale shark (Rhincodon typus), which was surprisingly found among both shark fin and gill plate samples. More than half of identified species fall under the IUCN Red List categories ‘Endangered’ and ‘Vulnerable’, raising further concerns about the impacts of this trade on the sustainability of these low productivity species.

Local environment rather than past climate determines community composition of mountain stream macroinvertebrates across Europe

Community assembly is determined by a combination of historical events and contemporary processes that are difficult to disentangle, but eco-evolutionary mechanisms may be uncovered by the joint analysis of species and genetic diversity across multiple sites. Mountain streams across Europe harbour highly diverse macroinvertebrate communities whose composition and turnover (replacement of taxa) among sites and regions remain poorly known. We studied whole-community biodiversity within and among six mountain regions along a latitudinal transect from Morocco to Scandinavia at three levels of taxonomic hierarchy: genus, species and haplotypes. Using DNA barcoding of four insect families (>3100 individuals, 118 species) across 62 streams, we found that measures of local and regional diversity and intraregional turnover generally declined slightly towards northern latitudes. However, at all hierarchical levels we found complete (haplotype) or high (species, genus) turnover among regions (and even among sites within regions), which counters the expectations of Pleistocene postglacial northward expansion from southern refugia. Species distributions were mostly correlated with environmental conditions, suggesting a strong role of lineage- or species-specific traits in determining local and latitudinal community composition, lineage diversification and phylogenetic community structure (e.g., loss of Coleoptera, but not Ephemeroptera, at northern sites). High intraspecific genetic structure within regions, even in northernmost sites, reflects species-specific dispersal and demographic histories and indicates postglacial migration from geographically scattered refugia, rather than from only southern areas. Overall, patterns were not strongly concordant across hierarchical levels, but consistent with the overriding influence of environmental factors determining community composition at the species and genus levels.

Uncovering unseen fungal diversity from plant DNA banks

Throughout the world DNA banks are used as storage repositories for genetic diversity of organisms ranging from plants to insects to mammals. Designed to preserve the genetic information for organisms of interest, these banks also indirectly preserve organisms’ associated microbiomes, including fungi associated with plant tissues. Studies of fungal biodiversity lag far behind those of macroorganisms, such as plants, and estimates of global fungal richness are still widely debated. Utilizing previously collected specimens to study patterns of fungal diversity could significantly increase our understanding of overall patterns of biodiversity from snapshots in time. Here, we investigated the fungi inhabiting the phylloplane among species of the endemic Hawaiian plant genus, Clermontia (Campanulaceae). Utilizing next generation DNA amplicon sequencing, we uncovered approximately 1,780 fungal operational taxonomic units from just 20 DNA bank samples collected throughout the main Hawaiian Islands. Using these historical samples, we tested the macroecological pattern of decreasing community similarity with decreasing geographic proximity. We found a significant distance decay pattern among Clermontia associated fungal communities. This study provides the first insights into elucidating patterns of microbial diversity through the use of DNA bank repository samples.

Scrutinizing key steps for reliable metabarcoding of environmental samples

Metabarcoding of environmental samples has many challenges and limitations that require carefully considered laboratory and analysis workflows to ensure reliable results. We explore how decisions regarding study design, laboratory set-up, and bioinformatic processing affect the final results, and provide guidelines for reliable study of environmental samples.

We evaluate the performance of four primer sets targeting COI and 16S regions characterizing arthropod diversity in bat faecal samples, and investigate how metabarcoding results are affected by parameters including: (1) number of PCR replicates per sample, (2) sequencing depth, (3) PCR replicate processing strategy (i.e. either additively, by combining the sequences obtained from the PCR replicates, or restrictively, by only retaining sequences that occur in multiple PCR replicates for each sample), (4) minimum copy number for sequences to be retained, (5) chimera removal, and (6) similarity thresholds for Operational Taxonomic Unit (OTU) clustering. Lastly, we measure within- and between-taxa dissimilarities when using sequences from public databases to determine the most appropriate thresholds for OTU clustering and taxonomy assignment.

Our results show that the use of multiple primer sets reduces taxonomic biases and increases taxonomic coverage. Taxonomic profiles resulting from each primer set are principally affected by how many PCR replicates are carried out per sample and how sequences are filtered across them, the sequence copy number threshold and the OTU clustering threshold. We also report considerable diversity differences between PCR replicates from each sample. Sequencing depth increases the dissimilarity between PCR replicates unless the bioinformatic strategies to remove allegedly artefactual sequences are adjusted according to the number of analysed sequences. Finally, we show that the appropriate identity thresholds for OTU clustering and taxonomy assignment differ between markers.

Metabarcoding of complex environmental samples ideally requires (1) investigation of whether more than one primer sets targeting the same taxonomic group is needed to offset primer biases, (2) more than one PCR replicate per sample, (3) bioinformatic processing of sequences that balance diversity detection with removal of artefactual sequences, and (4) empirical selection of OTU clustering and taxonomy assignment thresholds tailored to each marker and the obtained taxa.

Taxonomy assignment approach determines the efficiency of identification of OTUs in marine nematodes

Precision and reliability of barcode-based biodiversity assessment can be affected at several steps during acquisition and analysis of data. Identification of operational taxonomic units (OTUs) is one of the crucial steps in the process and can be accomplished using several different approaches, namely, alignment-based, probabilistic, tree-based and phylogeny-based. The number of identified sequences in the reference databases affects the precision of identification. This paper compares the identification of marine nematode OTUs using alignment-based, tree-based and phylogeny-based approaches. Because the nematode reference dataset is limited in its taxonomic scope, OTUs can only be assigned to higher taxonomic categories, families. The phylogeny-based approach using the evolutionary placement algorithm provided the largest number of positively assigned OTUs and was least affected by erroneous sequences and limitations of reference data, compared to alignment-based and tree-based approaches.

DNA in a bottle-Rapid metabarcoding survey for early alerts of invasive species in ports

Biota monitoring in ports is increasingly needed for biosecurity reasons and safeguarding marine biodiversity from biological invasion. Present and future international biosecurity directives can be accomplished only if the biota acquired by maritime traffic in ports is controlled. Methodologies for biota inventory are diverse and now rely principally on extensive and labor-intensive sampling along with taxonomic identification by experts. In this study, we employed an extremely simplified environmental DNA (eDNA) sampling methodology from only three 1-L bottles of water per port, followed by metabarcoding (high-throughput sequencing and DNA-based species identification) using 18S rDNA and Cytochrome oxidase I as genetic barcodes. Eight Bay of Biscay ports with available inventory of fouling invertebrates were employed as a case study. Despite minimal sampling efforts, three invasive invertebrates were detected: the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus and the polychaete Polydora triglanda. The same species have been previously found from visual and DNA barcoding (genetic identification of individuals) surveys in the same ports. The current costs of visual surveys, conventional DNA barcoding and this simplified metabarcoding protocol were compared. The results encourage the use of metabarcoding for early biosecurity alerts.

Close congruence between Barcode Index Numbers (bins) and species boundaries in the Erebidae (Lepidoptera: Noctuoidea) of the Iberian Peninsula

The DNA barcode reference library for Lepidoptera holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for conservation assessment programs. We gathered sequences for the barcode region of the mitochondrial cytochrome c oxidase subunit I gene from 160 of the 176 nominal species of Erebidae moths (Insecta: Lepidoptera) known from the Iberian Peninsula. These results arise from a research project which constructing a DNA barcode library for the insect species of Spain. New records for 271 specimens (122 species) are coupled with preexisting data for 38 species from the Iberian fauna. Mean interspecific distance was 12.1%, while the mean nearest neighbour divergence was 6.4%. All 160 species possessed diagnostic barcode sequences, but one pair of congeneric taxa (Eublemma rosea and Eublemma rietzi) were assigned to the same BIN. As well, intraspecific sequence divergences higher than 1.5% were detected in four species which likely represent species complexes. This study reinforces the effectiveness of DNA barcoding as a tool for monitoring biodiversity in particular geographical areas and the strong correspondence between sequence clusters delineated by BINs and species recognized through detailed taxonomic analysis.

Metabarcoding improves detection of eukaryotes from early biofouling communities: implications for pest monitoring and pathway management

In this experimental study the patterns in early marine biofouling communities and possible implications for surveillance and environmental management were explored using metabarcoding, viz. 18S ribosomal RNA gene barcoding in combination with high-throughput sequencing. The community structure of eukaryotic assemblages and the patterns of initial succession were assessed from settlement plates deployed in a busy port for one, five and 15 days. The metabarcoding results were verified with traditional morphological identification of taxa from selected experimental plates. Metabarcoding analysis identified > 400 taxa at a comparatively low taxonomic level and morphological analysis resulted in the detection of 25 taxa at varying levels of resolution. Despite the differences in resolution, data from both methods were consistent at high taxonomic levels and similar patterns in community shifts were observed. A high percentage of sequences belonging to genera known to contain non-indigenous species (NIS) were detected after exposure for only one day.

Online course on Metabarcoding

Its that time time of the year! In collaboration the the University of Guelph Open Ed department we are running another iteration of the distance education course on Metabarcoding taught by myself.

There are still spots available and the course will be running September 25 to October 20, 2017

This 4-week, web-based course provides an overview of the state of current technology and the various platforms used. The course consists of a series of online lectures and research exercises introducing different aspects of metabarcoding and environmental DNA research. I will also touch on the suite of bioinformatics tools available for sequence analysis and data interpretation.

We tried to cover as much as possible given the online format and the limited time participants usually have available to do such training. I am quite proud of it and feedback on last year's course was quite positive. The course is also designed with limited time resources of participants in mind. It usually takes an average of  four hours per week to go through the content and the materials. 

If you are interested there is still time to join. Sign up is here.

Weekend reads

Another busy week with no further posts. That should change next week as I have build quite a list of things to write about. Nevertheless, I wanted to ensure that I provide my weekly dose of interesting new reads. Here we go.

First molecular identification of the vertebrate hosts of Culicoides imicola in Europe and a review of its blood-feeding patterns worldwide: implications for the transmission of bluetongue disease and African horse sickness

Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife, livestock and, occasionally, humans. Culicoides imicola (Kieffer, 1913) is considered to be the main vector of the pathogens that cause bluetongue disease (BT) and African horse sickness (AHS) in southern Europe. The study of blood-feeding patterns in Culicoides is an essential step towards understanding the epidemiology of these pathogens. Molecular tools that increase the accuracy and sensitivity of traditional methods have been developed to identify the hosts of potential insect vectors. However, to the present group's knowledge, molecular studies that identify the hosts of C. imicola in Europe are lacking. The present study genetically characterizes the barcoding region of C. imicola trapped on farms in southern Spain and identifies its vertebrate hosts in the area. The report also reviews available information on the blood-feeding patterns of C. imicola worldwide. Culicoides imicola from Spain feed on blood of six mammals that include species known to be hosts of the BT and AHS viruses. This study provides evidence of the importance of livestock as sources of bloodmeals for C. imicola and the relevance of this species in the transmission of BT and AHS viruses in Europe.

Soil DNA metabarcoding and high-throughput sequencing as a forensic tool: considerations, potential limitations and recommendations

Analysis of physical evidence is typically a deciding factor in forensic casework by establishing what transpired at a scene or who was involved. Forensic geoscience is an emerging multi-disciplinary science that can offer significant benefits to forensic investigations. Soil is a powerful, nearly 'ideal' contact trace evidence, as it is highly individualistic, easy to characterise, has a high transfer and retention probability, and is often overlooked in attempts to conceal evidence. However, many real-life cases encounter close proximity soil samples or soils with low inorganic content, which cannot be easily discriminated based on current physical and chemical analysis techniques. The capability to improve forensic soil discrimination, and identify key indicator taxa from soil using the organic fraction is currently lacking. The development of new DNA sequencing technologies offers the ability to generate detailed genetic profiles from soils and enhance current forensic soil analyses. Here, we discuss the use of DNA metabarcoding combined with high-throughput sequencing (HTS) technology to distinguish between soils from different locations in a forensic context. Specifically, we provide recommendations for best practice, outline the potential limitations encountered in a forensic context and describe the future directions required to integrate soil DNA analysis into casework.

The detection of great crested newts year round via environmental DNA analysis

OBJECTIVE:

Analysis of environmental DNA (eDNA) is a method that has been used for the detection of various species within water bodies. The great crested newt (Triturus cristatus) has a short eDNA survey season (mid-April to June). Here we investigate whether this season could be extended into other months using the current methodology as stipulated by Natural England.

RESULTS:

Here we present data to show that in monthly water samples taken from two ponds (March 2014-February 2015) we were able to detect great crested newt DNA in all months in at least one of the ponds. Similar levels of great crested newt eDNA (i.e. highly positive identification) were detected through the months of March-August, suggesting it may be possible to extend the current survey window. In order to determine how applicable these observations are for ponds throughout the rest of the UK, further work in multiple other ponds over multiple seasons is suggested. Nevertheless, the current work clearly demonstrates, in two ponds, the efficacy and reproducibility of eDNA detection for determining the presence of great crested newts.

High occurrence of jellyfish predation by black-browed and Campbell albatross identified by DNA metabarcoding

Gelatinous zooplankton are a large component of the animal biomass in all marine environments, but are considered to be uncommon in the diet of most marine top predators. However, the diets of key predator groups like seabirds have conventionally been assessed from stomach content analyses, which cannot detect most gelatinous prey. As marine top predators are used to identify changes in the overall species composition of marine ecosystems, such biases in dietary assessment may impact our detection of important ecosystem regime shifts. We investigated albatross diet using DNA metabarcoding of scats to assess the prevalence of gelatinous zooplankton consumption by two albatross species, one of which is used as an indicator species for ecosystem monitoring. Black-browed and Campbell albatross scats were collected from eight breeding colonies covering the circumpolar range of these birds over two consecutive breeding seasons. Fish was the main dietary item at most sites, however cnidarian DNA, primarily from scyphozoan jellyfish was present in 42% of samples overall and up to 80% of samples at some sites. Jellyfish was detected during all breeding stages and consumed by adults and chicks. Trawl fishery catches of jellyfish near the Falkland Islands indicate a similar frequency of jellyfish occurrence in albatross diets in years of high and low jellyfish availability, suggesting jellyfish consumption may be selective rather than opportunistic. Warmer oceans and overfishing of finfish are predicted to favour jellyfish population increases and we demonstrate here that dietary DNA metabarcoding enables measurements of the contribution of gelatinous zooplankton to the diet of marine predators.

Revealing higher than expected meiofaunal diversity in Antarctic sediments: a metabarcoding approach

An increasing number of studies are showing that Antarctic mega- and macrofauna are highly diverse, however, little is known about meiofaunal biodiversity in sediment communities, which are a vital part of a healthy and functional ecosystem. This is the first study to analyse community DNA (targeting meiofauna) using metabarcoding to investigate biodiversity levels in sediment communities of the Antarctic Peninsula. The results show that almost all of the meiofaunal biodiversity in the benthic habitat has yet to be characterised, levels of biodiversity were higher than expected and similar to temperate regions, albeit with the existence of potentially new and locally adapted species never described before at the molecular level. The Rothera meiofaunal sample sites showed four dominant eukaryotic groups, the nematodes, arthropods, platyhelminthes, and the annelids; some of which could comprise species complexes. Comparisons with deep-sea data from the same region suggest little exchange of Operational Taxonomic Units (OTUs) between depths with the nematodes prevalent at all depths, but sharing the shallow water benthos with the copepods. This study provides a preliminary analysis of benthic Antarctic Peninsula meiofauna using high throughput sequencing which substantiates how little is known on the biodiversity of one of the most diverse, yet underexplored communities of the Antarctic: the benthos.

Weekend reads

It is Friday again - time for another batch of papers to read over the weekend. Thanks to a very active community there is never a shortage of studies to chose from.

Controls on eDNA movement in streams: Transport, Retention, and Resuspension

Advances in detection of genetic material from species in aquatic ecosystems, including environmental DNA (eDNA), have improved species monitoring and management. eDNA from target species can readily move in streams and rivers and the goal is to measure it, and with that infer where and how abundant species are, adding great value to delimiting species invasions, monitoring and protecting rare species, and estimating biodiversity. To date, we lack an integrated framework that identifies environmental factors that control eDNA movement in realistic, complex, and heterogeneous flowing waters. To this end, using an empirical approach and a simple conceptual model, we propose a framework of how eDNA is transported, retained, and resuspended in stream systems. Such an understanding of eDNA dispersal in streams will be essential for designing optimized sampling protocols and subsequently estimating biomass or organismal abundance. We also discuss guiding principles for more effective use of eDNA methods, highlighting the necessity of understanding these parameters for use in future predictive modeling of eDNA transport.

Nuclear genomes distinguish cryptic species suggested by their DNA barcodes and ecology

DNA sequencing brings another dimension to exploration of biodiversity, and large-scale mitochondrial DNA cytochrome oxidase I barcoding has exposed many potential new cryptic species. Here, we add complete nuclear genome sequencing to DNA barcoding, ecological distribution, natural history, and subtleties of adult color pattern and size to show that a widespread neotropical skipper butterfly known as Udranomia kikkawai (Weeks) comprises three different species in Costa Rica. Full-length barcodes obtained from all three century-old Venezuelan syntypes of U. kikkawai show that it is a rainforest species occurring from Costa Rica to Brazil. The two new species are Udranomia sallydaleyae Burns, a dry forest denizen occurring from Costa Rica to Mexico, and Udranomia tomdaleyi Burns, which occupies the junction between the rainforest and dry forest and currently is known only from Costa Rica. Whereas the three species are cryptic, differing but slightly in appearance, their complete nuclear genomes totaling 15 million aligned positions reveal significant differences consistent with their 0.00065-Mbp (million base pair) mitochondrial barcodes and their ecological diversification. DNA barcoding of tropical insects reared by a massive inventory suggests that the presence of cryptic species is a widespread phenomenon and that further studies will substantially increase current estimates of insect species richness.

Barcoding utility in a mega-diverse, cross-continental genus: keeping pace with Cyrtodactylus geckos

Over the past decade, DNA barcoding has become a staple of low-cost molecular systematic investigations. The availability of universal primers and subsidized sequencing projects (PolarBOL, SharkBOL, SpongeBOL) have driven this popularity, often without appropriate investigation into the utility of barcoding data for the taxonomic group of interest. Here, our primary aim is to determine the phylogenetic value of DNA barcoding (mitochondrial locus COI) within the gecko genus Cyrtodactylus. With >40 new species described since last systematic investigation, Cyrtodactylus represents one of the most diverse extant squamate genera, and their contemporary distribution spans the Indian subcontinent, eastward through Indochina, and into AustraloPapua. The complex biogeographic history of this group, and morphology-only designation of many species have complicated our phylogenetic understanding of Cyrtodactylus. To highlight the need for continued inclusive molecular assessment, we use Vietnamese Cyrtodactylus as a case study showing the geopolitically paraphyletic nature of their history. We compare COI to the legacy marker ND2, and discuss the value of COI as an interspecific marker, as well as its shortcomings at deeper evolutionary scales. We draw attention back to the Cold Code as a subsidized method for incorporating molecular methods into species descriptions in the effort to maintain accurate phylogenies.

Taxa-area relationship of aquatic fungi on deciduous leaves

One of the fundamental patterns in macroecology is the increase in the number of observed taxa with size of sampled area. For microbes, the shape of this relationship remains less clear. The current study assessed the diversity of aquatic fungi, by the traditional approach based on conidial morphology (captures reproducing aquatic hyphomycetes) and next generation sequencing (NGS; captures other fungi as well), on graded sizes of alder leaves (0.6 to 13.6 cm2). Leaves were submerged in two streams in geographically distant locations: the Oliveira Stream in Portugal and the Boss Brook in Canada. Decay rates of alder leaves and fungal sporulation rates did not differ between streams. Fungal biomass was higher in Boss Brook than in Oliveira Stream, and in both streams almost 100% of the reads belonged to active fungal taxa. In general, larger leaf areas tended to harbour more fungi, but these findings were not consistent between techniques. Morphospecies-based diversity increased with leaf area in Boss Brook, but not in Oliveira Stream; metabarcoding data showed an opposite trend. The higher resolution of metabarcoding resulted in steeper taxa-accumulation curves than morphospecies-based assessments (fungal conidia morphology). Fungal communities assessed by metabarcoding were spatially structured by leaf area in both streams. Metabarcoding promises greater resolution to assess biodiversity patterns in aquatic fungi and may be more accurate for assessing taxa-area relationships and local to global diversity ratios.

Small subunit ribosomal metabarcoding reveals extraordinary trypanosomatid diversity in Brazilian bats

BACKGROUND:

Bats are a highly successful, globally dispersed order of mammals that occupy a wide array of ecological niches. They are also intensely parasitized and implicated in multiple viral, bacterial and parasitic zoonosis. Trypanosomes are thought to be especially abundant and diverse in bats. In this study, we used 18S ribosomal RNA metabarcoding to probe bat trypanosome diversity in unprecedented detail.

METHODOLOGY/PRINCIPAL FINDINGS:

Total DNA was extracted from the blood of 90 bat individuals (17 species) captured along Atlantic Forest fragments of Espírito Santo state, southeast Brazil. The 18S ribosomal RNA was amplified by standard and/or nested PCR, then deep sequenced to recover and identify Operational Taxonomic Units (OTUs) for phylogenetic analysis. Blood samples from 34 bat individuals (13 species) tested positive for infection by 18S rRNA amplification. Amplicon sequences clustered to 14 OTUs, of which five identified as Trypanosoma cruzi I, T. cruzi III/V, Trypanosoma cruzi marinkellei, Trypanosoma rangeli, and Trypanosoma dionisii, and seven identified as novel genotypes monophyletic to basal T. cruzi clade types of the New World. Another OTU was identified as a trypanosome like those found in reptiles. Surprisingly, the remaining OTU was identified as Bodo saltans-closest non-parasitic relative of the trypanosomatid order. While three blood samples featured just one OTU (T. dionisii), all others resolved as mixed infections of up to eight OTUs.

CONCLUSIONS/SIGNIFICANCE:

This study demonstrates the utility of next-generation barcoding methods to screen parasite diversity in mammalian reservoir hosts. We exposed high rates of local bat parasitism by multiple trypanosome species, some known to cause fatal human disease, others non-pathogenic, novel or yet little understood. Our results highlight bats as a long-standing nexus among host-parasite interactions of multiple niches, sustained in part by opportunistic and incidental infections of consequence to evolutionary theory as much as to public health.

A look into a rumen

Roe Deer (Capreolus capreolus)

Dietary choices are central to our understanding of ecology and evolution. Still, many aspects of food choice have been hampered by time consuming procedures and methodological problems. Faster and cheaper methods, such as DNA metabarcoding, have therefore been widely adopted.

Traditional visual techniques based on morphology for diet analysis are often limited in their ability to identify all items found in a stomach. Unrecognizable content has to be ignored which can shift relative proportions within analysis. This happens mostly when digestion is well advanced, leaving researchers without any characters that would enable visual identification. It is well known that this can lead to biased results in studies of processes such as food web interactions and energy flow through an ecosystem. DNA barcoding has been suggested as a tool to overcome these issues and over the last 6-7 years a number of studies has been published all of which showed the increase of resolution by utilizing molecular techniques. 

A new study by Swedish researchers looked at the use of metabarcoding to assess ungulate diet through rumen content. They were interested in the diet of Fallow Deer (Dama dama) and Roe Deer (Capreolus capreolus).  In addition they set out to empirically compare metabarcoding to visual identifications. Not surprisingly, metabarcoding provided higher yield and precision of results although  they found that the methods were comparable, but they did not completely overlap. Sometimes the DNA method failed to identify food items that were found macroscopically...

When the colleagues applied both sets of data they found some very encouraging things.  In niche overlap analyses, presence/absence data showed that both methods came to very similar conclusions. When using the sequence count data and macroscopic weight, niche overlap was lower than when using presence-absence data yet tended to increase when using DNA compared to macroscopy. Nevertheless, the significant positive correlation between macroscopic quantity and number of DNA sequences counted from the same plant group give support for the use of metabarcoding to quantify plants in the rumen. 

Again, very promising evidence for the power of DNA barcoding in the context of dietary analysis. It would be very interesting to see how the results of this study correlate with an analysis of fecal samples. As a non-invasive method would increase sample size and effort quite extensively. The other suggestion I have is to use more than just one marker (here trnL). Include ITS or rbcL as well which might boost the metabarcoding success even more.

Biodiversity and climate warming

Meadow plots at Long-Term Ecological Research Station in Cedar Creek

Climate warming is predicted to alter species interactions, which could potentially lead to extinction events. However, there is an ongoing debate whether the effects of warming on biodiversity may be moderated by biodiversity itself.

A team of ecologists from the German Centre for Integrative Biodiversity Research (iDiv) and the University of Minnesota conducted long-running field experiments in 30 different meadow plots, some with only one plant species , and others with up to 16 different plant species. They warmed the meadows with heating lamps to approximately 3°C above ambient temperature. Subsequently, they recorded how this affected soil nematodes which were chosen for their important role for several ecosystem functions.

The colleagues found that warming can both increase and decrease biodiversity, and that the direction of the effect depends on how much biodiversity there is in the first place. After warming the monoculture plots the diversity of nematodes in those substantially declined. However, when the researchers warmed the plots with a high number of different plant species, the number of nematode species increased. However, those nematode species were also more closely related to each other. The reason was that these species had all been selected for a common characteristic, namely tolerance to a warmer environment. This increase in similarity can have implications for how well biological communities can respond to future environmental changes, potentially limiting the "insurance" effect inherent in a higher numbers of species.

The monoculture meadow created for the experiment resembled meadows found in intensively managed agricultural land. These new research findings therefore support conservationists who are advocating for more species-rich ecosystems and farmland to sustain biodiversity, in a warmer world. 

Weekend reads

Another week comes to an end and I have a few things I recommend reading. Enjoy!

Barcoding rotifer biodiversity in Mediterranean ponds using diapausing egg banks

The biodiversity of Mediterranean freshwater bodies is among the most threatened worldwide; therefore, its accurate estimation is an urgent issue. However, traditional methods are likely to underestimate freshwater zooplankton biodiversity due to its high species seasonality and cryptic diversity. We test the value of applying DNA barcoding to diapausing egg banks, in combination with the creation of a reference collection of DNA barcodes using adult individual samples, to characterize rotifer communities. We use monogonont rotifers from two lakes in Doñana National Park and one from Ruidera Natural Park in Spain as models to create a reference collection of DNA barcodes for taxonomically diagnosed adult individuals sampled from the water column, to compare with the sequences obtained from individual eggs from the diapausing egg banks. We apply two different approaches to carry out DNA taxonomy analyses, the generalized mixed Yule coalescent method (GMYC) and the Automatic Barcode Gap Discovery (ABGD), to the obtained sequences and to publicly available rotifer sequences. We obtained a total of 210 new rotifer COI sequences from all three locations (151 diapausing eggs and 59 adults). Both GMYC and ABGD generated the same 35 operational taxonomic units (OTUs), revealing four potential cryptic species. Most sequences obtained from diapausing eggs (85%) clustered with sequences obtained from morphologically diagnosed adults. Our approach, based on a single sediment sample, retrieved estimates of rotifer biodiversity higher than or similar to those of previous studies based on a number of seasonal samples. This study shows that DNA barcoding of diapausing egg banks is an effective aid to characterize rotifer diversity in Mediterranean freshwater bodies.

PCR-Based Method for the Detection of Toxic Mushrooms Causing Food-Poisoning Incidents

In this study, species-specific identification of five toxic mushrooms, Chlorophyllum molybdites, Gymnopilus junonius, Hypholoma fasciculare, Pleurocybella porrigens, and Tricholoma ustale, which have been involved in food-poisoning incidents in Japan, was investigated. Specific primer pairs targeting internal transcribed spacer (ITS) regions were designed for PCR detection. The specific amplicons were obtained from fresh, cooked, and simulated gastric fluid (SGF)-treated samples. No amplicons were detected from other mushrooms with similar morphology. Our method using one-step extraction of mushrooms allows rapid detection within 2.5 hr. It could be utilized for rapid identification or screening of toxic mushrooms.

The phylogeography of Myotis bat-associated rabies viruses across Canada

As rabies in carnivores is increasingly controlled throughout much of the Americas, bats are emerging as a significant source of rabies virus infection of humans and domestic animals. Knowledge of the bat species that maintain rabies is a crucial first step in reducing this public health problem. In North America, several bat species are known to be rabies virus reservoirs but the role of bats of the Myotis genus has been unclear due to the scarcity of laboratory confirmed cases and the challenges encountered in species identification of poorly preserved diagnostic submissions by morphological traits alone. This study has employed a collection of rabid bat specimens collected across Canada over a 25 year period to clearly define the role of particular Myotis species as rabies virus reservoirs. The virus was characterised by partial genome sequencing and host genetic barcoding, used to confirm species assignment of specimens, proved crucial to the identification of certain bat species as disease reservoirs. Several variants were associated with Myotis species limited in their Canadian range to the westernmost province of British Columbia while others were harboured by Myotis species that circulate across much of eastern and central Canada. All of these Myotis-associated viral variants, except for one, clustered as a monophyletic MYCAN clade, which has emerged from a lineage more broadly distributed across North America; in contrast one distinct variant, associated with the long-legged bat in Canada, represents a relatively recent host jump from a big brown bat reservoir. Together with evidence from South America, these findings demonstrate that rabies virus has emerged in the Myotis genus independently on multiple occasions and highlights the potential for emergence of new viral-host associations within this genus.

Applying pollen DNA metabarcoding to the study of plant-pollinator interactions

PREMISE OF THE STUDY:

To study pollination networks in a changing environment, we need accurate, high-throughput methods. Previous studies have shown that more highly resolved networks can be constructed by studying pollen loads taken from bees, relative to field observations. DNA metabarcoding potentially allows for faster and finer-scale taxonomic resolution of pollen compared to traditional approaches (e.g., light microscopy), but has not been applied to pollination networks.

METHODS:

We sampled pollen from 38 bee species collected in Florida from sites differing in forest management. We isolated DNA from pollen mixtures and sequenced rbcL and ITS2 gene regions from all mixtures in a single run on the Illumina MiSeq platform. We identified species from sequence data using comprehensive rbcL and ITS2 databases.

RESULTS:

We successfully built a proof-of-concept quantitative pollination network using pollen metabarcoding.

DISCUSSION:

Our work underscores that pollen metabarcoding is not quantitative but that quantitative networks can be constructed based on the number of interacting individuals. Due to the frequency of contamination and false positive reads, isolation and PCR negative controls should be used in every reaction. DNA metabarcoding has advantages in efficiency and resolution over microscopic identification of pollen, and we expect that it will have broad utility for future studies of plant-pollinator interactions.

Microbial Eukaryotes in an Arctic Under-Ice Spring Bloom North of Svalbard

Microbial eukaryotes can play prominent roles in the Arctic marine ecosystem, but their diversity and variability is not well known in the ice-covered ecosystems. We determined the community composition of microbial eukaryotes in an Arctic under-ice spring bloom north of Svalbard using metabarcoding of DNA and RNA from the hypervariable V4 region of 18S nrDNA. At the two stations studied, the photosynthetic biomass was dominated by protists >3 μm and was concentrated in the upper 70-80 m, above the thermocline and halocline. Hierarchical cluster analyses as well as ordination analyses showed a distinct clustering of the microbial eukaryote communities according to a combination of water mass and local environmental characteristics. While samples collected in the surface mixed layer differed distinctly between the two sites, the deeper communities collected in Atlantic Water were fairly similar despite being geographically distant. The differentiation of the microbial eukaryote communities of the upper mixed water was probably driven by local development and advection, while the lack of such differentiation in the communities of Atlantic Water reflects the homogenizing effect of water currents on microbial communities.

Biological annihilation

Catarina pupfish

No bells tolled when the last Catarina pupfish on Earth died. Newspapers didn't carry the story when the Christmas Island pipistrelle vanished forever.

Two vertebrate species go extinct every year on average, but few people notice, perhaps because the rate seems relatively slow - not a clear and present threat to the natural systems we depend on. This view overlooks trends of extreme decline in animal populations, which tell a more dire story with cascading consequences.

In a new publication researchers from Stanford University and UNAM in Mexico City draw a bleak picture of the future by talking about biological annihilation in an ongoing sixth mass extinction. The new study looks beyond species extinctions to provide a picture of dwindling populations and ranges. The colleagues mapped the ranges of 27,600 species of birds, amphibians, mammals and reptiles (nearly half of known terrestrial vertebrate species) and analyzed population losses in a sample of 177 well-studied mammal species between 1990 and 2015.

It turns out that more than 30% of vertebrate species are declining in population size and range. Of the subset mammals for which the researchers had detailed data, all have lost 30% or more of their geographic ranges and more than 40% of them have actually lost more than 80% of their ranges, most of which in southeast Asia. In general tropical regions have had the greatest number of decreasing species while temperate regions have seen similar or higher proportions of decreasing species. The study's mapping exercise suggests that as much as 50% of the number of animal individuals that once shared Earth have disappeared, as have billions of animal populations.

This is the case of a biological annihilation occurring globally, even if the species these populations belong to are still present somewhere on Earth... The massive loss of populations and species reflects our lack of empathy to all the wild species that have been our companions since our origins. It is a prelude to the disappearance of many more species and the decline of natural systems that make civilization possible.

What concerns me even more is the fact that this paper and many other studies exclusively look at vertrebrate species which are among the least diverse groups of animals. What about invertebrate diversity? Unfortunately, there is only anecdotal data and evidence. Humans haven't amassed much data to have a closer look but it is likely very similar if not worse. Few reliable data exist on the fate of important insect species. Scientists have tracked alarming declines in domesticated honey bees, monarch butterflies, and lightning bugs. But few have paid attention to the moths, hover flies, beetles, and countless other insects that buzz and flitter through the warm months. A recent article in Science gives a glimpse of what is going on in the invertebrate world. If we want to use such strong language as biological annihilation or erosion of biodiversity we might want to look a bit closer at the bugs around us even if the insights we gain might be even more disturbing that what we already learned from vertebrate studies.

Citizen science for butterfly conservation

Simplified butterfly assessment scheme used in Viel-Falter

Ordinary citizens have become increasingly important to scientific research over the past decade. Today, mobile phone technologies, relatively cheap cameras and almost ubiquitous internet connectivity have opened up new opportunities for conservation organisations to engage with ordinary citizens and encourage citizen science. A citizen scientist is a volunteer who collects and/or processes data as part of a scientific inquiry. This could mean noting the plants found on a day trip or more systematically recording wildlife in a special area.

Colleagues in Austria developed a specific assessment scheme suitable for citizen scientists and tested it at 35 sampling sites in Tyrol. They wanted to find out if trained and supervised school kids are able to systematically collect occurrence data on diurnal butterflies, and how the data could contribute to a permanent butterfly monitoring system. In 2013 they launched the citizen science project Viel-Falter.

By comparing with independent assessments conducted by professional butterfly experts, we investigated if the achieved data quality is sufficient to support a permanent butterfly monitoring system. Additionally, we investigated how the pupil’s motivation to engage in butterfly observation activities develops during the course of the project and what project factors might be crucial to support a continuous engagement.

Not surprisingly this comparison revealed that the degree of accordance varied substantially between different species or species groups. However, the data collected was successfully applied to predict the general habitat quality for butterflies using a linear regression model. This is indeed very good news as it shows that well-designed and executed citizen science projects can provide biodiversity data comparable to data gathered by professionals. Our experience during the School Malaise Project was very similar. 

There is an increased need and demand for large scale biodiversity assessments and continuous monitoring schemes but professional resources for collecting such data are rather limited. Citizen science projects like Viel-Falter might become a very important factor for future collecting and processing of biodiversity data. Such projects also come with immense added value as they generate authentic opportunities for environmental education.

Weekend reads

This short week passed by quickly. Nevertheless, I was able to find some interesting weekend reads for you.

Abrupt changes in the composition and function of fungal communities along an environmental gradient in the High Arctic

Fungi play a key role in soil-plant interactions, nutrient cycling, and carbon flow and are essential for the functioning of arctic terrestrial ecosystems. Some studies have shown that the composition of fungal communities is highly sensitive to variations in environmental conditions, but little is known about how the conditions control the role of fungal communities (i.e. their ecosystem function). We used DNA metabarcoding to compare taxonomic and functional composition of fungal communities along a gradient of environmental severity in Northeast Greenland. We analysed soil samples from fell fields, heaths, and snowbeds, three habitats with very contrasting abiotic conditions. We also assessed within-habitat differences by comparing three widespread microhabitats (patches with high cover of Dryas, Salix, or bare soil). The data suggest that, along the sampled mesotopographic gradient, the greatest differences in both fungal richness and community composition are observed among habitats, while the effect of microhabitat is weaker, although still significant. Furthermore, we found that richness and community composition of fungi are shaped primarily by abiotic factors and to a lesser, though still significant extent, by floristic composition. Along this mesotopographic gradient, environmental severity is strongly correlated with richness in all fungal functional groups: positively in saprotrophic, pathogenic, and lichenised fungi, and negatively in ectomycorrhizal and root-endophytic fungi. Our results suggest complex interactions amongst functional groups, possibly due to nutrient limitation or competitive exclusion, with potential implications on soil carbon stocks. These findings are important in light of the environmental changes predicted for the Arctic.

Predicting the ecological quality status of marine environments from eDNA metabarcoding data using supervised machine learning

Monitoring biodiversity is essential to assess the impacts of increasing anthropogenic activities in marine environments. Traditionally, marine biomonitoring involves the sorting and morphological identification of benthic macro-invertebrates, which is time-consuming and taxonomic-expertise demanding. High-throughput amplicon sequencing of environmental DNA (eDNA metabarcoding) represents a promising alternative for benthic monitoring. However, an important fraction of eDNA sequences remains unassigned or belong to taxa of unknown ecology, which prevent their use for assessing the ecological quality status. Here, we show that supervised machine learning (SML) can be used to build robust predictive models for benthic monitoring, regardless of the taxonomic assignment of eDNA sequences. We tested three SML approaches to assess the environmental impact of marine aquaculture using benthic foraminifera eDNA, a group of unicellular eukaryotes known to be good bioindicators, as features to infer macro-invertebrates based biotic indices. We found similar ecological status as obtained from macro-invertebrates inventories. We argue that SML approaches could overcome and even bypass the cost and time-demanding morpho-taxonomic approaches in future biomonitoring.

Temporal Dynamics of Soil Microbial Communities below the Seedbed under Two Contrasting Tillage Regimes

Agricultural productivity relies on a wide range of ecosystem services provided by the soil biota. Plowing is a fundamental component of conventional farming, but long-term detrimental effects such as soil erosion and loss of soil organic matter have been recognized. Moving towards more sustainable management practices such as reduced tillage or crop residue retention can reduce these detrimental effects, but will also influence structure and function of the soil microbiota with direct consequences for the associated ecosystem services. Although there is increasing evidence that different tillage regimes alter the soil microbiome, we have a limited understanding of the temporal dynamics of these effects. Here, we used high-throughput sequencing of bacterial and fungal ribosomal markers to explore changes in soil microbial community structure under two contrasting tillage regimes (conventional and reduced tillage) either with or without crop residue retention. Soil samples were collected over the growing season of two crops (Vicia faba and Triticum aestivum) below the seedbed (15-20 cm). Tillage, crop and growing stage were significant determinants of microbial community structure, but the impact of tillage showed only moderate temporal dependency. Whereas the tillage effect on soil bacteria showed some temporal dependency and became less strong at later growing stages, the tillage effect on soil fungi was more consistent over time. Crop residue retention had only a minor influence on the community. Six years after the conversion from conventional to reduced tillage, soil moisture contents and nutrient levels were significantly lower under reduced than under conventional tillage. These changes in edaphic properties were related to specific shifts in microbial community structure. Notably, bacterial groups featuring copiotrophic lifestyles or potentially carrying the ability to degrade more recalcitrant compounds were favored under conventional tillage, whereas taxa featuring more oligotrophic lifestyles were more abundant under reduced tillage. Our study found that, under the specific edaphic and climatic context of central Belgium, different tillage regimes created different ecological niches that select for different microbial lifestyles with potential consequences for the ecosystem services provided to the plants and their environment.

Whole mitochondrial genome capture from fecal samples and museum-preserved specimens

Population-scale molecular studies of endangered and cryptic species are often limited by access to high-quality samples. The use of non-invasively collected samples or museum-preserved specimens reduces the pressure on modern populations by removing the need to capture and handle live animals. However, endogenous DNA content in such samples is low, making shotgun sequencing a financially prohibitive approach. Here, we apply a target enrichment method to retrieve mitochondrial genomes from 65 museum specimens and 56 non-invasively collected fecal samples of two endangered great ape species, Grauer's gorilla and the eastern chimpanzee. We show that the applied method is suitable for a wide range of sample types that differ in endogenous DNA content, increasing the proportion of target reads to over 300-fold. By systematically evaluating biases introduced during target enrichment of pooled museum samples we show that capture is less efficient for fragments shorter or longer than the baits, that the proportion of human contaminating reads increases post-capture although capture efficiency is lower for human compared to gorilla fragments with a gorilla-generated bait, and that the rate of jumping PCR is considerable, but can be controlled for with a double-barcoding approach. We succeed in capturing complete mitochondrial genomes from fecal samples, but observe reduced capture efficiency as sequence divergence increases between the bait and target species. As previously shown for museum specimens, we demonstrate here that mitochondrial genome capture from field-collected fecal samples is a robust, and reliable approach for population-wide studies of non-model organisms.

So many trees

Why is tree biodiversity so large around the equator, moderate at mid-latitudes and monotonous at higher ones? As this is a global phenomenon most possible explanations involve long-term or large-scale mechanisms, such as climate stability (no glaciers in the tropics), rates of speciation (higher in the tropics) or rates of extinction (lower in the tropics according to the fossil record).

In order to explain the high tree species biodiversity in tropical rainforests Daniel Janzen and Joseph Connell independently suggested a very different mechanism that operates at a much smaller scale (conspecific negative density dependence - CNDD). They proposed that host-specific natural enemies which kill seeds and seedlings clumped near parent trees might keep locally common species from dominating a forest and give locally rare species space to flourish. This Janzen-Connell hypothesis is now nearly 50 years old, but it has been hard to evaluate, especially at the global scale. 

Utilizing the Smithsonian Center for Tropical Forest Science-Forest Global Earth Observatory (CTFS-ForestGEO) researchers now analyzed the data from 24 forest plots. Together these plots are home to more than 3,000 tree species and roughly 2.4 million trees. The analysis provided the first evidence that the Janzen-Connell effect contributes to the biodiversity gradient across tropical and temperate latitudes.

The results of the new study show that global patterns in tree species diversity reflect not only stronger CNDD at tropical versus temperate latitudes but also a latitudinal shift in the relationship between CNDD and species abundance. CNDD was stronger for rare species at tropical versus temperate latitudes, potentially causing the persistence of greater numbers of rare species in the tropics.

The key observation on which the Janzen-Connell hypothesis is based is that seedfall is heaviest under a parent tree but the young tend to do better away from their parent. In the tropics, this is stronger for rare species than for common ones. In the temperate zone rare and common species are equally affected, or in some cases it flips and becomes stronger for the common species than the rare ones. The colleagues find this result exciting because it may explain a puzzling characteristic of tropical forests - their diversity is due not to large numbers of species in general but rather to large numbers of rare species.

How can you pack more than a thousand species in a 50-hectare plot in the tropics if the rare species are being negatively impacted by these specialized enemies? You'd think that if these species are rare they'd be more likely to go extinct, so what maintains them in the system? ...Paradoxically, enemies can be beneficial, they kill, but by killing they prevent population booms and busts. If you have no enemies, you're going to have exponential population growth followed by a crash. If you add an enemy that tracks abundance, over time the population stabilizes. It's never going to become large, but the flip side is it's never going to crash. And so these enemies are a stabilizing force.

Weekend reads

A long weekend for Canadians lies ahead. Lots of parties around the country to celebrate Canada's 150th birthday. But the rest of the world has a regular weekend and all of you might want some good reads. Here we go:

Using high-throughput sequencing of ITS2 to describe Symbiodinium metacommunities in St. John, US Virgin Islands

Symbiotic microalgae (Symbiodinium spp.) strongly influence the performance and stress-tolerance of their coral hosts, making the analysis of Symbiodinium communities in corals (and metacommunities on reefs) advantageous for many aspects of coral reef research. High-throughput sequencing of ITS2 nrDNA offers unprecedented scale in describing these communities, yet high intragenomic variability at this locus complicates the resolution of biologically meaningful diversity. Here, we demonstrate that generating operational taxonomic units by clustering ITS2 sequences at 97% similarity within, but not across, samples collapses sequence diversity that is more likely to be intragenomic, while preserving diversity that is more likely interspecific. We utilize this 'within-sample clustering' to analyze Symbiodinium from ten host taxa on shallow reefs on the north and south shores of St. John, US Virgin Islands. While Symbiodinium communities did not differ between shores, metacommunity network analysis of host-symbiont associations revealed Symbiodinium lineages occupying 'dominant' and 'background' niches, and coral hosts that are more 'flexible' or 'specific' in their associations with Symbiodinium. These methods shed new light on important questions in coral symbiosis ecology, and demonstrate how application-specific bioinformatic pipelines can improve the analysis of metabarcoding data in microbial metacommunity studies.

Metabarcoding for the parallel identification of several hundred predators and their preys: application to bat species diet analysis (preprint)

Assessing diet variability between species, populations and individuals is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a two-step PCR protocol enabling the "all at once" taxonomic identification of bats and their arthropod preys for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed in triplicate. We compare the efficiency of DNA extraction methods and we evaluate the effectiveness of our protocol using the rate of bat identification success, taxonomic resolution, sensitivity, and amplification biases. Our strategy involves twice fewer steps than usually required in the other metabarcoding studies and reduces the probability to misassign preys to wrong samples. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. Our results illustrate the power of this approach to assess diet richness and variability within and between colonies. We therefore provide a rapid, resolutive and cost-effective screening tool for addressing evolutionary ecological issues or developing "chirosurveillance" and conservation strategies.

Short COI markers for freshwater macroinvertebrate metabarcoding (preprint)

Species diversity of metazoan bulk samples can be rapidly assessed using Cytochrome c oxidase I (COI) metabarcoding. However, in cases where only degraded DNA is available, e.g. from poorly conserved museum specimens, eDNA filtered from water or gut content analyses, universal primer sets that amplify only a short COI fragment are advantageous. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vivo by sequencing previously used freshwater macroinvertebrate mock communities of known composition and three monitoring samples from Romanian streams. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vivo , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2/BR2) revealed that efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

DNA Barcodes for the Northern European Tachinid Flies (Diptera: Tachinidae)

This data release provides COI barcodes for 366 species of parasitic flies (Diptera: Tachinidae), enabling the DNA based identification of the majority of northern European species and a large proportion of Palearctic genera, regardless of the developmental stage. The data will provide a tool for taxonomists and ecologists studying this ecologically important but challenging parasitoid family. A comparison of minimum distances between the nearest neighbors revealed the mean divergence of 5.52% that is approximately the same as observed earlier with comparable sampling in Lepidoptera, but clearly less than in Coleoptera. Full barcode-sharing was observed between 13 species pairs or triplets, equaling to 7.36% of all species. Delimitation based on Barcode Index Number (BIN) system was compared with traditional classification of species and interesting cases of possible species oversplits and cryptic diversity are discussed. Overall, DNA barcodes are effective in separating tachinid species and provide novel insight into the taxonomy of several genera.