A Concoction Pipeline for Generating Molecular Operational Taxonomic Units (MOTUs) Among Riparian and Aquatic Beetles

Biodiversity decline is transpiring at rates unimaginable, yet biodiversity monitoring is hampered by a plethora of factors, including the incomplete inventory of baseline diversity and the dwindling population of skilled taxonomists. Bridging this impediment in species inventory of "dark taxa", or hyperdiverse groups often understudied in taxonomy, is particularly crucial in the tropics facing insurmountable environmental pressures. While morphology-based alpha-taxonomy remains the gold standard in species identification, DNA-based methods have been showing tremendous potential in accelerating species delineation and discovery. Here, we describe a concoction pipeline for generating molecular operational taxonomic units (MOTUs) involving six molecular species delimitation approaches implemented on two highly inconspicuous beetle genera, namely Byrrhinus Motschulsky, 1858 of family Limnichidae and Anacaena Thomson, 1859 of family Hydrophilidae, from the Philippines. As this protocol focuses on processing DNA barcodes and given that these genetic data can be obtained either by sequencing or by downloading from online databases, the starting point for the protocol described here are the DNA sequences. Given the COI barcodes, the pipeline utilizes a combination of three threshold-based molecular species delimitation approaches, namely TaxonDNA, K2P, and ASAP, which use sequence alignment as input data. Additionally, the pipeline proceeds with three coalescent-based approaches, namely PTP-ML, PTP-BI, and mPTP, which require a tree file as input data. Some approaches entail the use of web servers, while others utilize local software. With the selection of algorithmic methods presented, MOTUs can be identified, whether by consensus or by majority. Remarkably, this pipeline delineated almost conspicuous beetle species from the Philippines, even those documented from the same or adjacent localities.