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In the spring of 2007, DEPTHX was deployed in Zacat\u00f3n, a deep (\u223c318\u2009m), limestone, phreatic sinkhole (cenote) in northeastern Mexico. As DEPTHX descended, it generated a 3-D map based on the processing of range data from 54 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. Traditional 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 Zacat\u00f3n, and collected biological samples based on an autonomous decision, which revealed novel microbial diversity in a previously unexplored environment. 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