*3.2. Mitochondrial Processing in C. vulgaris Resembles That Seen in Embryophytes.*

We also analyzed the mRNA termini of the mitochondrial mRNAs of *C. vulgaris* and found the 5 and 3-UTRs to be much longer and more variable than those observed in

*P. duplex* and *C. reinhardtii*. We also detected non-template polyadenylation of 3 termini, the possibility of RNA secondary structures in processed 3- UTRs, and the possibility of t-elements downstream of some 3 termini. The *C. vulgaris* chondriome is one of the smaller among charophytic algae, 67,737 bp, with intergenic region sizes more similar to Chlorophyeae than higher plants, yet with gene content, gene synteny, and intron placement very similar to the much larger bryophyte chondriomes [62,63]. After post-transcriptional processing, we found that the 3 and 5- UTR lengths in *C. vulgaris* were more similar to those found in embryophytes than chlorophytes. In embryophytes, 5 termini are of varying lengths, ranging from dozens to thousands of nucleotides in length, presumably due to a stepwise 5 maturation process [29–36,82–86]. Among embryophytes, 3- UTR lengths tend to be more consistent in length and mature mRNAs lack non-template oligonucleotide additions other than polyadenylation, which marks the mRNA for degradation [44–47]. Embryophyte mitochondria utilize tRNAs and RNA secondary structure (t-elements) to guide endonucleolytic cleavage [31–33]. The presence of relatively long 5 and 3- UTRs, the polyadenylation of some 3 termini, the possibility that secondary structures may guide the formation of some 3 termini, and the possibility that secondary structures may form in the 3- UTRs of *C. vulgaris* mitochondrial mRNAs to presumably provide a target for 3- specific RPFs demonstrates that their RNA processing is more similar to embryophytes than chlorophytes. One recent proteomic survey of *Arabidopsis* mitochondria estimated that 14.9% of the nuclear encoded mitochondrial-targeted proteome is devoted to mRNA processing [87]. Our results suggest that the RNA processing mechanisms and perhaps the large numbers of nuclear-encoded RPFs that locate to the mitochondria in higher plants were present in the common ancestor of all streptophytes.

In *C. vulgaris*, circularized full length coding regions were detected for five genes and only fragments were found for four other genes. Unlike *P. duplex*, there were no repeat motifs associated with the ligation sites and no polyA additions detected in the circularized transcripts. Among embryophyte mitochondria, circularized RNAs were characterized in *Hordeum vulgare* and *Arabidopsis*, but all of the examples were fragmented coding regions [88,89]. Our data suggest that the circularization of mRNAs in *C. vulgaris* may be a hybrid of chlorophytes and embryophytes.
