Biology subjects

Elbrecht, V.

Publications and source records attributed to Elbrecht, V..

7 recordsLinked to original sources

bioRxiv2020

Effects of Malaise trap spacing on species richness and composition of terrestrial arthropod bulk samples

The Malaise trap has gained popularity for assessing diverse terrestrial arthropod communities because it collects large samples with modest effort. A number of factors that influence collection efficiency, placement being one of them. For instance, when designing larger biotic surveys using arrays of Malaise traps we need to know the optimal distance between individual traps that maximises observable species richness and community composition. We examined the influence of spacing between Malaise traps by metabarcoding samples from two field experiments at a site in Waterloo, Ontario, Canada. For one experiment, we used two trap pairs deployed at weekly increasing distance (3m increments from 3 to 27 m). The second experiment involved a total of 10 traps set up in a row at 3m distance intervals for three consecutive weeks. Results show that community similarity of samples decreases over distance between traps. The amount of species shared between trap pairs shows drops considerably at about 15m trap-to-trap distance. This change can be observed across all major taxonomic groups and for two different habitat types (grassland and forest). Large numbers of OTUs found only once within samples cause rather large dissimilarity between distance pairs even at close proximity. This could be caused by a large number of transient species from adjacent habitat which arrive at the trap through passive transport, as well as capture of rare taxa, which end up in different traps by chance.

ecology↗

bioRxiv2020

Pooling size sorted malaise trap fractions to maximise taxon recovery with metabarcoding

O_LISmall and rare specimens can remain undetected when metabarcoding bulk samples with a high size heterogeneity of specimens. This is especially critical for malaise trap samples, where most of the biodiversity is often contributed by small specimens. How to size sort and in which proportions to pool these samples has not been widely explored. We set out to find a size sorting strategy that maximizes taxonomic recovery but remains highly scalable and time efficient. C_LIO_LIThree 3 malaise trap samples where size sorted into 4 size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate samples never sorted, pooled in equal proportions and in 4 different proportions favoring the small size fractions. DNA from the pooled fractions as well as the individual size classes were extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally wet sieving strategies were explored. C_LIO_LIThe small size fractions harbored the highest diversity, and were best represented when pooling in favor of small specimens. Not size sorting a sample leads to a 45-77% decrease in taxon recovery compared to size sorted samples. A size separation into only 2 fractions (below 4 mm and above) can already double taxon recovery compared to not sorting. However, increasing the sequencing depth 3-4 fold can also increase taxon recovery to comparable levels, but remains biased toward biomass rich taxa in the sample. C_LIO_LIWe demonstrate that size fractionizing bulk malaise samples can increase taxon recovery. The most practical approach is wet sieving into two size fractions, and proportional pooling of the lysates in favor of the small size fraction (80-90% volume). However, in large projects with time constraints, increasing sequencing depth can also be an alternative solution. C_LI

molecular biology↗

bioRxiv2020

A workflow for accurate metabarcoding using nanopore MinION sequencing

Metabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short-read high-throughput sequencing platforms. The Oxford Nanopore MinION, a portable sequencing platform, represents a low-cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%. To test if the MinION represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full-length consensus DNA barcodes (658bp of cytochrome oxidase I - COI) for a bulk mock sample of 50 aquatic invertebrate species. By applying two different laboratory protocols, we generated two MinION runs that were used to build consensus sequences. We also developed a novel Python pipeline, ASHURE, for processing, consensus building, clustering, and taxonomic assignment of the resulting reads. We were able to show that it is possible to reduce error rates to a median accuracy of up to 99.3% for long RCA fragments (>45 barcodes). Our pipeline successfully identified all 50 species in the mock community and exhibited comparable sensitivity and accuracy to MiSeq. The use of RCA was integral for increasing consensus accuracy, but it was also the most time-consuming step during the laboratory workflow and most RCA reads were skewed towards a shorter read length range with a median RCA fragment length of up to 1262bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding but we recommend the exploration of other isothermal amplification procedures to improve consensus length.

bioinformatics↗

bioRxiv2020

Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

DNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database. However, when universal primers are used on environmental DNA (eDNA) isolated from stream water, macroinvertebrate read and OTU numbers are typically "watered down", i.e. diluted, compared to whole specimen bulk samples due to greater co-amplification of abundant non-target taxa such as algae and bacteria. Because stream macroinvertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.

ecology↗

bioRxiv2019

Metabarcoding unsorted kick-samples facilitates macroinvertebrate-based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA

Many studies have highlighted the potential of DNA-based methods for the biomonitoring of freshwater macroinvertebrates, however only a few studies have investigated homogenisation of bulk samples that include debris to reduce sample-processing time. In order to explore the use of DNA-based methods in water quality assessment in South Africa, this study compares morphological and molecular-based identification of freshwater macroinvertebrates at the mixed higher taxon and mOTU level while investigating abundance and comparing mOTU recovery with historical species records. From seven sites across three rivers in South Africa, we collected a biomonitoring sample, an intensive-search comprehensive sample and an eDNA sample per site. The biomonitoring sample was picked and scored according to standard protocols and the leftover debris and comprehensive samples were homogenised including all debris. DNA-based methods recovered higher diversity than morphology, but did not always recover the same taxa, even at the family level. Regardless of the differences in taxon scores, most DNA-based methods except some eDNA samples, returned the same water quality assessment category as the standard morphology-based assessment. Homogenised comprehensive samples recovered more freshwater invertebrate diversity than all other methods. The eDNA samples recovered 2 to 10 times more mOTUs than any other method, however 90% of reads were non-target and as a result eDNA recovered the lowest target diversity. However, eDNA did find some target taxa that the other methods failed to detect. This study shows that unsorted samples recover the same water quality scores as a morphology-based assessment and much higher diversity scores than both picked and eDNA samples. As a result, there is potential to integrate DNA-based approaches into existing metrics quickly while providing much more information for the development of more refined metrics at the species or mOTU level with distributional data which can be used for conservation and biodiversity management.

ecology↗

bioRxiv2019

Enhancing DNA metabarcoding performance and applicability with bait capture enrichment and DNA from conservative ethanol

Metabarcoding is often presented as an alternative identification tool to compensate for coarse taxonomic resolution and misidentification encountered with traditional morphological approaches. However, metabarcoding comes with two major impediments which slow down its adoption. First, the picking and destruction of organisms for DNA extraction are time and cost consuming and do not allow organism conservation for further evaluations. Second, current metabarcoding protocols include a PCR enrichment step which induces errors in the estimation of species diversity and relative biomasses. In this study, we first evaluated the capacity of capture enrichment to replace PCR enrichment using controlled freshwater macrozoobenthos mock communities. Then, we tested if DNA extracted from the fixative ethanol (etDNA) of the same mock communities can be used as an alternative to DNA extracted from pools of whole organisms (bulk DNA). We show that capture enrichment provides more reliable and accurate representation of species occurrences and relative biomasses in comparison with PCR enrichment for bulk DNA. While etDNA does not permit to estimate relative biomasses, etDNA and bulk DNA provide equivalent species detection rates. Thanks to its robustness to mismatches, capture enrichment is already an efficient alternative to PCR enrichment for metabarcoding and, if coupled to etDNA, is a time-saver option in studies where presence information only is sufficient.

molecular biology↗

bioRxiv2018

Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

DNA metabarcoding is an attractive approach for monitoring biodiversity. However, it is subject to biases that often impede detection of all species in a sample. In particular, the proportion of sequences recovered from each species depends on its biomass, mitome copy number, and primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 BINs, a species proxy. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body partitions (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicon generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, other variables did. As expected, the best recovery was obtained from the Single Leg treatment, but the Bulk Abdomen produced a more uniform read abundance than the Bulk Leg or Composite Leg samples. Primer choice also influenced species recovery. Our results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to obtain reproducible results and increase its cost-effectiveness.

ecology↗