magna ~28 kb capuana piriformis capuana magna piriformis parva

for studying organelle transcription from some of the most poorly studied but ecologically important organisms on Earth. One group elected for transcriptome sequencing by the MMETSP was Polytomella—a monophyletic green algal genus of free-living, freshwater, non-photosynthetic unicells, closely related to Chlamydomonas reinhardtii Nakada et al., 2008; Smith et al., 2013 . There are presently four known Polytomella lineages, repre- sented by Polytomella piriformis, P. parva, P. capuana, and P. magna. The mtDNAs of these four species have been studied in great detail Fig. 1 A Fan and Lee, 2002; Smith and Lee, 2008; Smith et al., 2010, 2013 . They are small 13–28 kb, made up of 1–2 linear chromosomes with invertedly repeated telomeres, have matching or very similar gene orders, which are organized into opposing transcriptional units, and contain identical gene contents, encoding 7 standard mitochondrial proteins, 1 tRNA, and 2 rRNAs. Moreover, in P. magna, the deepest-branching member of the genus, every gene in the mitochondrial genome is duplicated and in an inverted orientation relative to its partner Fig. 1 A Smith et al., 2013 . Together, these unusual genomic features make Polytomella an excellent candidate for studying organelle gene expression. Here, we use Illumina RNA-Seq to examine the mitochondrial transcriptomes of the four known Polytomella lineages. Our goal is to unravel the roles, if any, of the Polytomella mitochondrial telomeres and palindromic elements in gene expression, and to characterize aspects of posttranscriptional processing. In a broader sense, we want to explore the effectiveness of RNA-Seq for recov- ering entire mitochondrial genomes. In the end, we find many ben- efits as well as some limitations to using RNA-Seq for organelle chr1: ~3 kb nad5 nad5 1 1 nad1 nad1 nad6 nad6 cob cob 5 4 6 3 2 4 4 2 3 6 4 5 7 7 cox1 cox1 nad4 nad4 nad2 nad2 1 1 8 8 2 2 3 3 TIR TIR 1 2 3 4 5 6 7 8 9 TIR cob 1 nad6 7 cox1 nad4 nad2 8 2 3 nad5 4 2 3 6 4 5 nad1 TIR 1 TIR cob 1 nad6 7 cox1 nad4 nad2 8 2 3 nad5 4 2 3 6 4 5 nad1 TIR 1 TIR TIR P. magna ~28 kb P. capuana ~13 kb chr2: ~13 kb

P. piriformis

10 protein-coding tRNA Met SSU rRNA-coding 4 fragments LSU rRNA-coding 8 fragments telomere A Polytomella mitochondrial genomic and transcriptomic architectures Photosynthetic | Nonphotosynthetic P. capuana P. magna P. piriformis P. parva

C. reinhardtii B Tree of Polytomella spp.