COI Leray fragment of adult and juvenile spiders

Biodiversity loss is a major problem at the global scale, so bioinventories and monitoring samplings are essential to identify and tackle threats to biodiversity. Arthropods are usually misrepresented in these inventories, despite being the largest component of animal diversity. This is mainly due to the difficulty of identification in some arthropod groups, such as spiders, which can only be morphologically identified to species level at adult stages. Thus, juvenile spiders are usually discarded during sorting despite representing around half of the specimens, but the impact this has on community-level diversity estimates is still unknown.

Here, we use a metabarcoding approach to assess the contribution that juvenile stages of spiders collected across the Iberian Peninsula have on diversity estimates, and their effect on diversity patterns across communities. We also compare the proportion of reads and the abundance of each family in the samples to assess the quantitative power of metabarcoding.

The total number of species obtained as adults and juveniles was 363 and 331, respectively, with species found exclusively as juveniles representing an increase of 35% with respect to those found as adults. We observed that the differences in community composition between spider communities, especially the differences between communities at different latitudes, are greatly reduced when information about immature stages is taken into account. The proportion of reads obtained from metabarcoding for certain spider families was positively related to their proportion in weight and abundance in the sample, although the intensity of this relation was not constant across families. This suggests that metabarcoding data are to a certain extent quantitative.

These findings do not question the information provided by adult-based inventories, but add a novel yet relevant layer of knowledge previously overlooked that may influence some of the interpretations derived from biological inventories.

Methods
For adult spiders:

Legs of spiders were used for DNA extraction (more information about the specimens in Crespo et al, 2018). Total genomic DNA was extracted using the REDExtract-N-Amp™ Tissue PCR Kit Protocol from Sigma-Aldrich, following the manufacturer’s protocol and performed in 96 well-plates. We amplified the mitochondrial cytochrome c oxidase (COI). The LCOI1490/ HCO2198 was the preferred combination of primers, while Nancy was used as a replacement for HCOI2198 and Ron as replacement for LCOI1490 (in that order). For problematic amplifications, we used the internal primers mlCOIintF/jgHCOI2198. The polymerase chain reaction (PCR) was performed in 96-well plates using 8 µl REDExtract-N-Amp™ PCR ReadyMix from Sigma-Aldrich, primers forward and reverse, 4 µl of diluted DNA and ultrapure, distilled water up to a total reaction volume of 20 µl. PCR products were cycle-sequenced in both directions at Macrogen Inc. (Seoul, South Korea). We trimmed the sequences to keep only the Leray 313 bp fragment.

For juvenile spiders:

We homogenised all the collected immature spiders with the help of liquid nitrogen. We obtained two extraction replicates from each homogenised plot sample, and we extracted a fraction of 0.3 g from each replicate using a PowerSoil DNA Isolation Kit (QIAGEN, Valencia, CA, USA). We used Leray-XT primers to amplify the Leray 313 bp fragment of mitochondrial cytochrome c oxidase (COI) and sequenced it in an Illumina Miseq V3 2x250 bp. OBITools were used for paired-end assembly, demultiplexing and quality control. Chimeras were removed with uchime_denovo in VSearch. Swarm 2.0 with d=13 was used for clustering MOTUs. Ecotag was used for taxonomic assignment using custom reference databases for the COI Leray region. LULU was used for removing supernumerary MOTUs arising from pseudogenes. Singleton MOTUs were removed.