Novel Microbial Diversity Retrieved by Autonomous Robotic Exploration of the World's Deepest Vertical Phreatic Sinkhole

The deep phreatic thermal explorer (DEPTHX)is an autonomous underwater vehicle designed to navigate an unexplored environment, generate high-resolution three-dimensional (3-D)maps, collect biological samples based on an autonomous sampling decision, and return to its origin. In the spring of 2007, DEPTHX was deployed in Zacato´n, a deep (*318m), limestone, phreatic sinkhole (cenote)in northeastern Mexico. As DEPTHX descended, it generated a 3-D map based on the processing of range data from54 onboard sonars. The vehicle collected water column samples and wall biomat samples throughout the depth profile of the cenote. Post-expedition sample analysis via comparative analysis of 16S rRNA gene sequences revealed a wealth of microbial diversity. Tradi-tional Sanger gene sequencing combined with a barcoded-amplicon pyrosequencing approach revealed novel, phylum-level lineages from the domains Bacteria and Archaea; in addition, several novel subphylum lineages were also identified. Overall, DEPTHX successfully navigated and mapped Zacato´n, and collected biological samples based on an autonomous decision, which revealed novel microbial diversity in a previously unexplored environment.