Biodiversity detection

DNA metabarcoding has been proven to be a very effective and powerful tool to study arthropod communities as a whole by overcoming the taxonomic impediment, allowing detection of a large variety of taxa (see Liu et al., 2020 and references therein). To the best of our knowledge, this is the first time that such a wide biodiversity analysis has been carried out at the species level in Mediterranean evergreen oak woodlands (“dehesas” or “montados”). Previous studies on soil arthropod communities from this habitat had been performed by grouping at higher taxonomic levels: Class, Order and, in rarer cases, Family (Andrés et al., 1999; Rota et al., 2015; Sadaka & Ponge, 2003). At a lower taxonomic level, only checklists of species from certain families have been reported (e.g. Zaballos, 1983), but in no case related to the effects of livestock on arthropods. In the present study we could analyse it at the MOTU level, despite not all of them could be assigned to Linnean species.
Identification success differed among taxonomic levels depending on the availability of reference sequences. Almost all arthropod MOTUs were identified to Order level, and more than 80% to Family level. We could thus identify most of the arthropod families coexisting in these Iberian holm oak savannas. However, identification success at the species level was lower: we could assign Linnean species names to only 40% of the MOTUs. This highlights the importance of promoting classical taxonomy research as the solid base to create comprehensive and reliable reference databases of DNA barcodes, indispensable for the use of molecular identification methods. Moreover, these databases should be geographically unbiased, since in the Mediterranean region, where our study took place, reference barcodes are scarcer than, for example, in Central Europe, which decreases identification likelihood (Gaytán et al., 2020).
The use of two different genetic markers increased biodiversity detection success. The complementary use of 16S in addition to COI did not contribute detection at the Order level, but it did at the Family level. Although more families were exclusively detected by COI, others were only retrieved by 16S. Moreover, these families may have had an important role in the biodiversity analysis, as differences between treatments in the richness at MOTU level were always more pronounced using the 16S dataset. These results agree with a previous study which concluded that the best markers to detect Hexapoda subphylum barcodes are found in the mitochondrial ribosomal RNA (rRNA) genes, such as 12S and 16S (Marquina et al., 2019b). However, our results suggest that their conclusions may be applicable not just on Hexapoda, but also to all Arthropoda phylum. Nevertheless, the lack of reference data compared to COI ballasts the use of this marker if the main aim is species identification.
For this work, we chose a non-destructive approach for the DNA extraction from the bulk samples, as we were interested in keeping the individual arthropods for further morphological analysis. A series of recent studies have tested the suitability of these non-destructive DNA extraction protocols, finding them as a promising alternative to sample homogenization in terms of biodiversity detection (Batovska et al., 2021; Iwaszkiewicz-Eggebrecht et al., 2023; Marquina et al., 2022; Nielsen et al., 2019). However, there may be limitations relevant for the analysis of our samples: for instance, both Iwaszkiewicz-Eggebrecht et al. (2023) and Marquina et al. (2022) found that ants (Hymenoptera: Formicidae) were detected in their mock communities in much lower proportion and occasions than other taxa. This could have affected our results, as we worked with ground-dwelling arthropod communities, in which ants are prominent members. Nonetheless, we did find a great diversity of MOTUs belonging to Formicidae (18 with COI and 12 with 16S), which we consider acceptable.