DNA metabarcoding for biodiversity monitoring in a national park: Screening for invasive and pest species

Hardulak, Laura A.; Morinière, Jérôme; Hausmann, Axel; Hendrich, Lars; Schmidt, Stefan; Doczkal, Dieter; Müller, Jörg; Haszprunar, Gerhard

doi:10.1111/1755-0998.13212

Cited by 36 publications

(35 citation statements)

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“…Aside from eDNA (environmental DNA) metabarcoding, where free extracellular DNA is processed (e.g. from soil, water, faeces), DNA can be extracted from enclosed communities (cDNA), more precisely, the sample’s fixative ethanol (Batovska et al, 2021; Hajibabaei et al, 2012; Martins et al, 2019; Zizka et al, 2019) or propylene glycol (Martoni et al, 2021), from added lysis buffer (Giebner et al, 2020; Ji et al, 2013; Kirse et al,2021) or from homogenised tissue of specimens (Hardulak et al, 2020; Mata et al, 2020; Zizka et al, 2020). While the latter approach is currently considered most effective to assess biodiversity pattern (Hardulak et al, 2020; Marquina et al, 2019; Persaud et al, 2021; Zenker et al, 2020; Zizka et al, 2019) it prevents subsequent morphological determinations (Nielsen et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…from soil, water, faeces), DNA can be extracted from enclosed communities (cDNA), more precisely, the sample’s fixative ethanol (Batovska et al, 2021; Hajibabaei et al, 2012; Martins et al, 2019; Zizka et al, 2019) or propylene glycol (Martoni et al, 2021), from added lysis buffer (Giebner et al, 2020; Ji et al, 2013; Kirse et al,2021) or from homogenised tissue of specimens (Hardulak et al, 2020; Mata et al, 2020; Zizka et al, 2020). While the latter approach is currently considered most effective to assess biodiversity pattern (Hardulak et al, 2020; Marquina et al, 2019; Persaud et al, 2021; Zenker et al, 2020; Zizka et al, 2019) it prevents subsequent morphological determinations (Nielsen et al, 2019). Homogenisation and tissue-based DNA extraction can be conducted from wet (Beentjes et al, 2019; Gibson et al, 2015; Porter et al, 2019) samples in ethanol or from dried tissue after ethanol evaporation (Elbrecht et al, 2019; Hardulak et al, 2020; Hausmann et al, 2020; Steinke et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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Recommendations for tissue homogenisation and extraction in DNA metabarcoding of Malaise trap samples

Zizka

Geiger

Hörren³

et al. 2022

Preprint

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With increased application of DNA metabarcoding in fast and high-resolution biodiversity assessment, various laboratory protocols have been optimised in recent years and their further evaluation is subject of current research. Homogenisation of bulk samples and subsequent DNA extraction from destructed tissue is one way of starting the metabarcoding process. This essential step in the protocol can either be conducted from wet sample material (e.g. bulk insect samples) soaked in fixative or from completely dried individuals. While the latter method appears to produce more consistent results, it is time consuming and more prone to cross-contamination. We tested both homogenisation approaches with regard to time efficiency and biodiversity assessment of complex arthropod bulk samples, in particular how the amount of processed tissue affects taxon recovery. Both approaches reveal similar taxa compositions and detect a similar total OTU diversity in a single extraction reaction. Increased amounts of tissue used in DNA extraction improved OTU diversity detection and recovered particularly specific low-biomass taxa, making this approach valuable for samples with high biomass and/or diversity. Due to less handling time and lower vulnerability for cross-contamination we recommend the processing of wet material when sample homogenisation is applied.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Recommendations for tissue homogenisation and extraction in DNA metabarcoding of Malaise trap samples

Zizka

Geiger

Hörren³

et al. 2022

Preprint

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“…DNA metabarcoding is a useful approach to characterize arthropod communities. Instead of DNA barcoding individual specimens (Hebert et al, 2003), DNA is usually extracted from homogenized bulk samples (Zizka, Geiger & Leese, 2020;Mata et al, 2020;Turunen et al, 2021;Hardulak et al, 2020). A barcoding marker is amplified for the whole community and sequenced with high throughput sequencing.…”

Section: Introductionmentioning

confidence: 99%

“…In fact, most non-marine studies do metabarcode complete bulk samples without prior size sorting (e.g. Hardulak et al, 2020;Hausmann et al, 2020;Steinke et al, 2020;Bush et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Pooling size sorted Malaise trap fractions to maximize taxon recovery with metabarcoding

Elbrecht

Bourlat

Hörren³

et al. 2021

PeerJ

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Background Small and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient. Methods Three Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored. Results The small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample. Conclusion We demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80–90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.

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“…DNA metabarcoding thus considerably up-scales diversity assessments of bulk samples, and accuracy is continuously improved through the refinement of molecular approaches and the expansion of reference libraries (BOLD, Ratnasingham and Hebert 2007;GBOL, Geiger et al 2016a). Results from German Malaise trapping programs prove the strength of this approach for biodiversity assessments (Morinière et al 2016;Geiger et al 2016b;Hausmann et al 2020;Hardulak et al 2020).…”

Section: The Project Dina-diversity Of Insects In Nature Protected Areasmentioning

confidence: 94%

Diversity of Insects in Nature protected Areas (DINA): an interdisciplinary German research project

et al. 2021

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Insect declines and biodiversity loss have attracted much attention in recent years, but lack of comprehensive data, conflicting interests among stakeholders and insufficient policy guidance hinder progress in preserving biodiversity. The project DINA (Diversity of Insects in Nature protected Areas) investigates insect communities in 21 nature reserves in Germany. All selected conservation sites border arable land, with agricultural practices assumed to influence insect populations. We taught citizen scientists how to manage Malaise traps for insect collection, and subsequently used a DNA metabarcoding approach for species identification. Vegetation surveys, plant metabarcoding as well as geospatial and ecotoxicological analyses will help to unravel contributing factors for the deterioration of insect communities. As a pioneering research project in this field, DINA includes a transdisciplinary dialogue involving relevant stakeholders such as local authorities, policymakers, and farmers, which aims at a shared understanding of conservation goals and action pathways. Stakeholder engagement combined with scientific results will support the development of sound policy recommendations to improve legal frameworks, landscape planning, land use, and conservation strategies. With this transdisciplinary approach, we aim to provide the background knowledge to implement policy strategies that will halt further decline of insects in German protected areas.

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DNA metabarcoding for biodiversity monitoring in a national park: Screening for invasive and pest species

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 36 publications

References 102 publications

Recommendations for tissue homogenisation and extraction in DNA metabarcoding of Malaise trap samples

Recommendations for tissue homogenisation and extraction in DNA metabarcoding of Malaise trap samples

Pooling size sorted Malaise trap fractions to maximize taxon recovery with metabarcoding

Diversity of Insects in Nature protected Areas (DINA): an interdisciplinary German research project

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