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Background: Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data. Methodology/Principal Findings: Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection. Conclusions/Significance: Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This phylogenetic framework is applicable to understanding the evolution of specific carotenoid biosynthetic proteins or the unique characteristics of carotenoid biosynthetic evolution in a specific phylogenetic lineage. Together, these analyses suggest a ‘‘bramble’’ model for microbial carotenoid biosynthesis whereby later biosynthetic steps exhibit greater evolutionary plasticity and reticulation compared to those closer to the biosynthetic ‘‘root’’. Structural diversification may be constrained (‘‘trimmed’’) where selection is strong, but less so where selection is weaker. These analyses also highlight likely productive avenues for future research and bioprospecting by identifying both gaps in current knowledge and taxa which may particularly facilitate carotenoid diversification.

The high-throughput annotation of open reading frames (ORFs) required by modern genome sequencing projects necessitates computational protocols that sometimes annotate orthologous ORFs inconsistently. Such inconsistencies hinder comparative analyses by non-uniformly extending or truncating 5′ and/or 3′ sequence ends, causing ORFs that are in fact identical to artificially diverge. Whereas strategies exist to correct such inconsistencies during whole-genome annotation, equivalent software designed to correct subsets of these data without genome reannotation is lacking. We therefore developed ORFcor, which corrects annotation inconsistencies using consensus start and stop positions derived from sets of closely related orthologs. ORFcor corrects inconsistent ORF annotations in diverse test datasets with specificities and sensitivities approaching 100% when sufficiently related orthologs (e.g., from the same taxonomic family) are available for comparison. The ORFcor package is implemented in Perl, multithreaded to handle large datasets, includes related scripts to facilitate high-throughput phylogenomic analyses, and is freely available at www.currielab.wisc.edu/downloads.html.