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The chloroplast is one of two organelles containing a separate genome that codes for essential and distinct cellular functions such as photosynthesis. Given the importance of chloroplasts in plant metabolism, the genomic architecture and gene content have been strongly conserved through long periods of time and as such are useful molecular tools for evolutionary inferences. At present, complete chloroplast genomes from over 4000 species have been deposited into publicly accessible databases. Despite the large number of complete chloroplast genomes, comprehensive analyses regarding genome architecture and gene content have not been conducted for many lineages with complete species sampling. In this study, we employed the genus  Populus  to assess how more comprehensively sampled chloroplast genome analyses can be used in understanding chloroplast evolution in a broadly studied lineage of angiosperms. We conducted comparative analyses across  Populus  in order to elucidate variation in key genome features such as genome size, gene number, gene content, repeat type and number, SSR (Simple Sequence Repeat) abundance, and boundary positioning between the four main units of the genome. We found that some genome annotations were variable across the genus owing in part from errors in assembly or data checking and from this provided corrected annotations. We also employed complete chloroplast genomes for phylogenetic analyses including the dating of divergence times throughout the genus. Lastly, we utilized re-sequencing data to describe the variations of pan-chloroplast genomes at the population level for  P .  euphratica . The analyses used in this paper provide a blueprint for the types of analyses that can be conducted with publicly available chloroplast genomes as well as methods for building upon existing datasets to improve evolutionary inference.

Chloroplast genomes in Populus (Salicaceae): comparisons from an intensively sampled genus reveal dynamic patterns of evolution