Strengthening marine amphipod DNA barcode libraries for environmental monitoring

Environmental DNA barcoding technology is gaining innovative applications when monitoring diverse tropics of marine environment. Since previous studies have shown that most marine eDNA sequences cannot be assigned to any species and in most cases to any phyla, since only 15 percent of the animal species is barcoded and accessible in reference libraries, we propose to test the effectiveness of current DNA barcode reference libraries in identifying amphipod barcodes and/or strengthening the existing library. Over 2500 amphipod individuals were collected for morphological analysis and DNA barcoding from estuarine sediments. We barcoded 22 amphipod species belonging to 17 genera, 13 families in the order Amphipoda. We found that 13 species (59 percent) were missing from the reference barcode libraries and were first time barcoded. More than 80 percent of the species were not reported previously form the sampled area. The minimum and maximum inter-specific pair-wise distance values was found to be respectively 0.16 and 5.51 percent. We show that defining family specific species threshold values would be imperative, rather than expecting a universal barcode gap for amphipod species. The overall mean pair-wise distance, nucleotide diversity and Tajima statistics were 3.59 percent, 0.27 and 2.62, respectively. Species identification through neighbour joining tree shows that the taxa of same species clustered together indicating the efficacy of COI sequences in amphipod species delineation. There is a strong need to increase the number of amphipod species barcodes in the reference database, because 59 percent of barcoded species were found absent. For better facilitation of environmental monitoring, the sequences were published via public databases. We show that with the advent of next generation sequencing technologies, reference datasets like ours will become important for health evaluation and monitoring of different environments using amphipod barcodes.