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DNA-based species identification techniques

Working towards Integrative TaxonomY.

In general, this term refers to DNA, RNA or protein-based techniques used for species identification. In wild bee biodiversity research, the ‘molecular component’ of integrative taxonomy often refers to DNA-based identification methods such as DNA barcoding and metabarcoding.

“Species delineation is a long-time process during which specialists acquire expertise in a taxonomic group and aggregate evidence of various kinds (morphological, molecular, ecological, etc.). DNA- based identification methods are not a threat to taxonomists but rather a necessary component to make hypotheses and decisions about species limits, without aiming to provide final conclusions on taxa description.” (Cilia et al. 2025)

Barcoding is a molecular technique that allows for DNA-based species identification. To determine the identity of an unknown organism, researchers compare the sequence of a short fragment of DNA from a standardized region of the genome against a reference database. In animals, a segment of mitochondrial DNA (the region of the cytochrome c oxidase I or “COI” gene) is most commonly used for this purpose. In the last two decades, this approach has been used to characterize the molecular diversity of wild bees worldwide, contributing significantly to the available taxonomic knowledge of this pollinator group.

Identification is the very first step for conservation biology projects. Determining which species exist in the area of interest (What is living here?) is fundamental to answer more elaborated questions (What is new?, What is being lost?) and to propose efficient conservation strategies. Traditionally, the description and identification of insect species is carried out by taxonomic experts working in national research institutions (e.g. natural history museums, universities, etc.). To address the current biodiversity crisis, this task is more important than ever. However, since there is a shortage of experienced taxonomists in Luxembourg who can provide this information, traditional identification techniques have been supported with other approaches to achieve the volume of data required for proper biodiversity assessment.

In these efforts, DNA-based species identification techniques are a powerful ally. If a DNA sample is available, these methods can identify many wild bees to genus or species level, even if the user lacks specialised taxonomic knowledge. They are also capable of distinguishing cryptic or hard to identify sister species, regardless of sex or life stage, and they can potentially identify even incomplete specimens. In addition, online platforms have made it possible to compare sequences of wild bees from Luxembourg with references from all over the world, thus overcoming important geographical restrictions.

Therefore, by combining traditional practices with alternative identification techniques, it is possible to increase the amount of data available for conservation purposes and ecological studies, as well as to support efforts in species discovery and identification.

Animal DNA barcoding: Step by Step

1.

Tissue sampling

The process starts by collecting a small tissue sample or body part (e.g. hair, blood, saliva, exoskeleton, etc) from the organism of interest. The DNA is contained within the cells that form the tissue.

2.

DNA extraction

In this step, chemicals are used to separate and “clean up” the DNA from all other components of the tissue sample (e.g. proteins, oils, fats, etc). Depending on the type of sample, different techniques can be used, but all of them have one goal in common: to isolate DNA from all interfering substances so it can be properly amplified.

3.

Amplification

Once the DNA has been cleaned, the target gene fragment is amplified using polymerase chain reaction (PCR). PCR is a type of in vitro DNA replication that generates copies of the target fragment under controlled conditions. read more

4.

Sequencing

In order to identify an unknown organism, it is key to determine the specific nucleotide sequence of the amplified DNA molecules using a laboratory technique called “Sequencing”. read more

5.

Comparison against reference

The sequence of the genetic marker obtained in the previous step (the “DNA barcode”) is then algorithmically compared against a reference database. DNA-based techniques, such as barcoding, crucially rely on reference sequences and the accuracy of their results depends directly on the quality of these references. Without a library of validated DNA barcodes to compare against, unknown sequences cannot be identified with precision. read more

DNA barcoding in a nutshell

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MNHNL barcoding projects

Local and international efforts are currently underway to fill DNA barcoding data gaps for the European wild bee fauna.
To support these initiatives, all new barcodes produced by the beelibre project have been uploaded to the online barcode collection of the MNHNL. At the Public Data Portal of BOLD Systems, you can access an overview of all deposited DNA barcodes of the museum (search code: “MNHNL”), as well as those produced by partner institutions and research centers abroad.

References

Cilia, G., Ranalli, R., Zavatta, L., & Flaminio, S (2024). Hidden and Wild: An Integrated Study of European Wild Bees. Springer, Switzerland:1-528. https://doi.org/10.1007/978-3-031-76742-5

Herrera-Mesías F, Ep Jarboui IK, Weigand AM (2022) A metabarcoding framework for wild bee assessment in Luxembourg. Journal of Hymenoptera Research 94: 215–246. https://doi.org/10.3897/jhr.94.84617

Herrera Mesías F, Weigand AM (2021) Updates to the checklist of the wild bee fauna of Luxembourg as inferred from revised natural history collection data and fieldwork. Biodiversity Data Journal 9: e64027. https://doi.org/10.3897/BDJ.9.e64027

Hochkirch, A., Casino, A., Penev, L., Allen, D., Tilley, L., Georgiev, T., Gospodinov, K. and Barov, B. 2022. European Red List of Insect Taxonomists. Luxembourg: Publication Office of the European Union.

Reverté, S., Miliˇcic, M., Aˇcanski, J., Andric, A., Aracil, A., Aubert, M. et al. (2023) National records of 3000 European bee and hoverfly species: A contribution to pollinator conservation. Insect Conservation and Diversity, 16(6), 758–775. Available from: https://doi.org/10.1111/icad. 12680

Schmidt, S., Schmid‐Egger, C., Morinière, J., Haszprunar, G., & Hebert, P. D. (2015). DNA barcoding largely supports 250 years of classical taxonomy: identifications for Central European bees (Hymenoptera, Apoidea partim). Molecular Ecology Resources, 15(4), 985-1000.