SCIENCE GOALS: WHEN RECONSTRUCTING EVOLUTIONARY HISTORIES OF LIFE ON EARTH PRIMARY SOURCES OF INFORMATION ARE GEOLOGIC DATA APPLICABLE MOSTLY TO PHANEROZOIC LIFE FORMS AND MOLECULAR DATA FOR PRE-CAMBRIAN LIFE. TIMELINES INFERRED FROM THESE TWO SOURCES ARE GENERALLY IN AGREEMENT BUT MAJOR CHALLENGES FOR KEY EVOLUTIONARY STEPS REMAIN. THIS IS ESPECIALLY TRUE FOR TIMELINES OF EARLY MICROBIAL LIFE THAT DUE TO THE ALMOST COMPLETE LACK OF GEOLOGIC RECORD ARE HEAVILY DEPENDENT ON MOLECULAR-BASED APPROACHES THAT OFTEN PRODUCE CONTRASTING EVOLUTIONARY HISTORIES. TO IMPROVE THE CONFIDENCE IN THE ESTIMATED TIMELINES IT IS THEREFORE IMPERATIVE TO (I) RECONCILE CONTRASTING TIMELINES FROM MOLECULAR DATA WITH EACH OTHER AND ALSO WITH THE GEOLOGIC TIMELINE AND (II) DESIGN AN OBJECTIVE PROCEDURE TO IDENTIFY BIASES IN ESTIMATIONS EVEN IN CASES WHEN ALTERNATIVE INDEPENDENT EVIDENCE IS LACKING. OBJECTIVES: IN RESPONSE TO THESE NEEDS HERE WE PROPOSE TO ESTABLISH AN OBJECTIVE AND COMPUTATIONALLY EASY PROCEDURE TO EVALUATE THE ACCURACY OF PUBLISHED AND FUTURE TIMETREES THAT WILL LEAD TO MORE ACCURATE AND CONCORDANT TIMELINES. OUR METHOD INVESTIGATES THE PARAMETERS ASSOCIATED WITH CALIBRATION POINTS AND EVOLUTIONARY RATE VARIATION AMONG LINEAGES PARAMETERS THAT ARE KNOWN TO AFFECT THE ACCURACY OF RELAXED MOLECULAR CLOCKS. WE PROPOSE TO USE A RELATIVE FRAMEWORK ESTABLISHED FROM PRIOR-FREE MODELS (FOURTH GENERATION MOLECULAR CLOCKS) TO DETECT BIASES IN THESE PARAMETERS WHICH CAN THEN BE REMOVED TO INCREASE ACCURACY OF INDIVIDUAL ESTIMATES. WE SHOW PRELIMINARY DATA FROM THE APPLICATION OF THE PROPOSED APPROACH IN SIMULATED SCENARIOS AND AN EMPIRICAL DATASET RELATED TO A HIGHLY CONTROVERSIAL EVOLUTIONARY EVENT THE TIMELINE OF THE ORIGIN AND DIVERSIFICATION OF ANIMALS. THE RESULTS SUPPORT THE VALIDITY OF THE PROPOSED APPROACH THAT WE PLAN TO APPLY TO HUNDREDS OF PUBLISHED TIMETREES WITH KNOWN DISAGREEMENTS IN AN EFFORT TO PRODUCE AN ACCURATE TIMELINE OF THE EVOLUTION OF LIFE. THE ESTABLISHED PROCEDURE WILL BE PARTICULARLY USEFUL IN INVESTIGATIONS OF EARLY MICROBIAL LIFE FOR WHICH MOLECULAR DATA IS VIRTUALLY THE ONLY RECORD OF THEIR EVOLUTIONARY HISTORY. METHODS: BASED ON THE OVERALL GOAL OF THIS PROPOSAL WE HAVE IDENTIFIED TWO AIMS. IN AIM #1 WE WILL FOCUS ON THE EVALUATION OF CALIBRATION BOUNDARIES AND THEIR EFFECTS ON DIVERGENCE TIMES. THIS IS AN APPLIED AIM THAT WILL FIRST EVALUATE TIMETREES FROM THE LITERATURE TO IDENTIFY THE MOST COMMON WEAKNESSES AND RE-ESTIMATE NEW TIMETREES WITH IMPROVED PARAMETERS AND TAXONOMIC SAMPLING. THE OUTCOMES OF THIS AIM WILL BE THE FIRST LARGE-SCALE ASSESSMENT OF PUBLISHED TIMETREES AND THE ESTIMATION OF NEW MORE ACCURATE DIVERGENCE TIMES FOR A WIDE RANGE OF TAXONOMIC GROUPS FOCUSING ON EARLY MICROBIAL SPECIES. AIM #2 WILL PROVIDE THEORETICAL AND COMPUTATIONAL SUPPORT FOR THE APPLIED ANALYSIS OF THE PREVIOUS AIM BY INVESTIGATING DISTRIBUTIONS OF RATE VARIATION AMONG LINEAGES FOR DIFFERENT SIMULATED DATASETS. THESE DISTRIBUTIONS WILL SERVE AS REFERENCE TO DETERMINE PATTERNS OF RATE VARIATION IN EMPIRICAL DATA AND WILL PROVIDE INFORMATION TO OPTIMIZE OUR METHODOLOGY. THE NEW TESTING PROCEDURE DEVELOPED IN AIMS #1 AND #2 WILL THEN BE IMPLEMENTED IN A NEW SOFTWARE THAT WILL ALLOW EXPERTS AND NON-EXPERTS TO EVALUATE THE DEPENDENCY OF THEIR ESTIMATED DIVERGENCE TIMES ON THE CHOSEN PARAMETERS. SIGNIFICANCE: WE EXPECT THIS PROJECT TO BE HIGHLY SIGNIFICANT BECAUSE BY IMPROVING MOLECULAR DATING METHODS IT WILL PROVIDE THE MEANS TO ESTIMATE A TIMELINE OF THE EVOLUTION OF LIFE THAT WILL BRING INTO CLOSER AGREEMENT BIOLOGICAL GEOLOGICAL AND PALEONTOLOGICAL VIEWS OF THE CONDITIONS ON EARLY EARTH. RECONSTRUCTING A CHRONOLOGY OF LIFE ON EARTH HAS BEEN A LONGSTANDING GOAL IN ASTROBIOLOGY AS IT EMPOWERS RESEARCHERS TO DRAW INFERENCES ON THE CO-EVOLUTION OF LIFE AND HABITABLE CONDITIONS OF A PLANET.