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Background  Chlorophyceae is one of three most species-rich green algal classes and also the only class in core Chlorophyta whose monophyly remains uncontested as gene and taxon sampling improves. However, some key relationships within Chlorophyceae are less clear-cut and warrant further investigation. The present study combined genome-scale chloroplast data and rich sampling in an attempt to resolve the ordinal classification in Chlorophyceae. The traditional division into Sphaeropleales and Volvocales (SV), and a clade containing Oedogoniales, Chaetopeltidales, and Chaetophorales (OCC) was of particular interest with the addition of deeply branching members of these groups, as well as the placement of several  incertae sedis  taxa.    Methods  We sequenced 18 chloroplast genomes across Chlorophyceae to compile a data set of 58 protein-coding genes of a total of 68 chlorophycean taxa. We analyzed the concatenated nucleotide and amino acid datasets in the Bayesian and Maximum Likelihood frameworks, supplemented by analyses to examine potential discordant signal among genes. We also examined gene presence and absence data across Chlorophyceae.    Results  Concatenated analyses yielded at least two well-supported phylogenies: nucleotide data supported the traditional classification with the inclusion of the enigmatic Treubarinia into Sphaeropleales  sensu lato . However, amino acid data yielded equally strong support for Sphaeropleaceae as sister to Volvocales, with the rest of the taxa traditionally classified in Sphaeropleales in a separate clade, and Treubarinia as sister to all of the above. Single-gene and other supplementary analyses indicated that the data have low phylogenetic signal at these critical nodes. Major clades were supported by genomic structural features such as gene losses and trans-spliced intron insertions in the plastome.    Discussion  While the sequence and gene order data support the deep split between the SV and OCC lineages, multiple phylogenetic hypotheses are possible for Sphaeropleales  s.l . Given this uncertainty as well as the higher-taxonomic disorder seen in other algal groups, dwelling on well-defined, strongly supported Linnaean orders is not currently practical in Chlorophyceae and a less formal clade system may be more useful in the foreseeable future. For example, we identify two strongly and unequivocally supported clades: Treubarinia and Scenedesminia, as well as other smaller groups that could serve a practical purpose as named clades. This system does not preclude future establishment of new orders, or emendment of the current ordinal classification if new data support such conclusions.

Order, please! Uncertainty in the ordinal-level classification of Chlorophyceae