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DNA barcoding is an alternative method to the traditional morphological taxonomic classification, and has frequently been used to identify species of aquatic macroinvertebrates (generally considered those large enough to be seen without magnification). Many are crucial indicator organisms in the bioassessment of freshwater (e.g.: Ephemeroptera, Plecoptera, Trichoptera) and marine (e.g. Annelida, Echinoderms, Molluscs) ecosystems.
Since its introduction, the field of DNA barcoding has matured to bridge the gap between traditional taxonomy and molecular systematics. This technique has the ability to provide more detailed taxonomic information, particularly for cryptic, small, or rare species. DNA barcoding involves specific targeting of gene regions that are found and conserved in most animal species, but have high variation between members of different species. Accurate diagnosis depends on low intraspecific variation compared with that between species, a short DNA sequence such as Cytochrome Subunit Oxidase I gene (COI), would allow precise allocation of an individual to a taxon.
While the concept of using DNA sequence divergence for species discrimination has been reported earlier, Hebert et al. (2003) were pioneers in proposing standardization of DNA barcoding as a method of molecularly distinguishing species.[1]
Specimens collection for DNA barcoding does not differ from the traditional methods, apart from the fact that the samples should be preserved in high concentration (>70%) ethanol.[2] It has been indicated that the typical protocol of storing benthic samples in formalin has an adverse effect on DNA integrity.[3]
The key concept for barcoding macroinvertebrates, is proper selection of DNA markers (DNA barcode region) to amplify appropriate gene regions, using PCR techniques. The DNA barcode region needs to be ideally conserved within a species, but variable among different (even closely related) species and therefore, its sequence should serve as a species-specific genetic tag. Therefore, the selection of the marker plays an important role.[4] Cytochrome Subunit Oxidase I gene (COI) is one of the most widely used markers in barcoding of macroinvertebrates. Other markers that can be used are ribosomal RNA genes 16S and 18S.
Moreover, sorting invertebrates into different size categories is useful, since specimens in a sample can vary widely in biomass, depending on species and life stage.[5]
For further details on methods see DNA barcoding.
Main article: Metabarcoding |
Due to the significant number of taxa that compose aquatic macroinvertebrate communities, DNA metabarcoding method is generally used to assess distinct taxa within bulk or water samples. DNA metabarcoding is a method that consists of the same workflow as DNA barcoding, distinguished by the use of high-throughput sequencing (HTS) technologies. The potential of DNA metabarcoding in the assessment and monitoring of various taxonomic groups, has been successfully demonstrated in several studies.[6][7] Numerous researchers have used metabarcoding methods to classify benthic macroinvertebrates from tissue samples,[8] indicating its feasibility and higher sensitivity from classical taxonomy methods. Others, validate the use of next-generation sequencing (NGS) technologies in environmental samples to evaluate water quality in marine ecosystems[9] and in freshwater biodiversity studies,[10] including macroinvertebrate species assessment. Applications of these technologies in environmental samples is constantly increasing.[11] Most of the recent studies are based on advancing eDNA approaches' implementation, field validation, platform and barcode choice or database limitations.[12]
Macroinvertebrates (meta)barcoding methods are often used in:
There are also many challenges when it comes to genetic barcoding of aquatic macroinvertebrates: