Table 2.

Overview of the bioinformatic workflow applied, including the use each custom software script, names and version numbers of third-party software tools employed or initiated by the scripts, and the computation time required to perform each automated analysis step. Computation time was measured on a machine with an i5-2500K 3.3 GHz Intel Quad-Core processor (Santa Clara, CA, USA), 24 GB of RAM and a Linux 4.14.1 kernel and is given as the average computation time for each of the three newly sequenced plastid genomes. Abbreviations: min = minutes; n.a. = not applicable; s = seconds.

Analysis Step	Details of Analysis	Custom Software Script	Third-Party Software Tool Employed 1	Computation Time (Mean)
Quality control of reads	Generating ordered intersection of R1/R2 reads	Script 01	bioawk v.20110810	6 min 29 s
	Filtering of reads by quality score	Script 02	FASTX Toolkit v.0.0.14	14 min 03 s
Genome assembly	Assembling reads to contigs	Script 03	IOGA v.20160910 Python v.2.7.14	17 min 12 s
	Stitching contigs to complete genomes	Manual step	Geneious v.10.2.3
Evaluation of assembly	Confirming the IR boundaries	Script 04	BLAST+ v.2.4.0	<1 s
	Extracting reads that map to final assembly	Script 05	bowtie2 v.2.3.2 samtools v.1.7 BEDtools v.2.27.1	3 min 37 s
	Generating assembly statistics	Script 06	bowtie2 v.2.3.2 samtools v.1.7 BEDtools v.2.27.1	7 min 20 s
Genome annotation	Generating raw annotations	Manual step	DOGMA cpGAVAS
	Converting annotations of DOGMA to GFF format	Script 07	n.a.	<1 s
	Combining annotations of DOGMA and cpGAVAS	Script 08	Python v.2.7.14 Perl v.5.26.1	<1 s
Evaluation of annotations	Confirming the validity of annotations	Manual step	Geneious v.10.2.3
Sequence alignment	Extraction and alignment of coding regions	Script 09	Python v.2.7.14	39 s
	Removal of gap positions	Script 10	statAl v.1.4.rev22 trimAl v.1.4.rev22	<1 s
Phylogenetic analysis	Phylogenetic tree inference under ML, including bootstrapping	Script 11	R v.3.4.4	17 s

¹ Software tools such as sed, grep and awk, which are part of most Unix command shells, are not listed separately. Similarly, individual R, Python or Perl dependencies are not listed separately.