Table 1.

Table of all currently available Florideophyceae mitochondrion genomes that were examined in this study with GenBank Accession, genome length, and A/T%

Species	GenBank Accession	Length	AT Content (%)	Reported Missing Genes	Notes
Ah. plicata	KF649303	32,878	66.6	atp8, rpl20	atp8: PCR of atp8 region shows gene is present (KX687876). Previously published assembly was missing a single nucleotide, which resulted in a pseudogene. rpl20: With ATA start codon, gene is fully present (243 bp), but ATG start codon gene is short (147 bp). Translations of both align with other red algal rpl20.
A. taxiformis	KJ398158	26,097	73.3	sdhD	sdhD: Annotated in GenBank as ‘gene/hypothetical protein CDS’, BlastP of translated sequence hits other red algal sdhD sequences and aligns well with other translated red algal sdhD genes.
Ce. japonicum	KJ398159	26,200	71.5	atp4 (as ymf39), sdhC, sdhD, TatC (as secY), rpl20	atp4: Annotated in GenBank as ‘gene/hypothetical protein CDS’, BlastP of translated sequence hits other red algal atp4 (ATP synthase B chain precursor) sequences and aligns well with other translated red algal atp4 (ymf39) genes. In Yang et al. (2015), considered atp4, this is a case of nomenclature causing confusion. sdhC: In the correct location there is a region annotated as ‘gene/hypothetical protein CDS’, but it is shorter than other Florideophyceae sdhC genes. Translation shows conservation of residues, particularly at 5’ region of the sequence. Possibly truncated at 3’ end, resequencing would help clarify. sdhD: Found in published data, but was unannotated 16,636>16,403. TatC: Seems to be the result of homopolymer sequence error, though possibility of pseudogene remains. Homology based on translation alignment of region from 23,168>23,590. rpl20: Likely an actual loss, all sequenced members of Ceramiales also missing rpl20. Truncated from 5’ end and about half the gene remains as a pseudogene.
Ce. sungminbooi	KU145004 and KU145005	24,508	71.2	noted as partial in article (Hughey and Boo 2016)	rpl20: Likely a loss, all sequenced members of Ceramiales also missing rpl20. Truncated from 5’ end and about half the gene remains as a pseudogene. sdhC: Found, as unannotated 10,977 > 10,678 in conserved location between atp9 and sdhB in KU145004. sdhD: Found, as unannotated ORF 15,284 > 15,036 in conserved location between nad4 and nad2 in KU145004. TatC: Found, as unannotated ORF 15,277 > 15,029 in conserved location between nad4 and nad2 in KU145004.
		24,494	71.2
Ch. crispus	NC_001677	25,836	72.1	—	Complete
C. polysiphoniae	KX687877	25,357	79.4	rpl20	rpl20: Likely an actual loss, all sequenced members of Ceramiales also missing rpl20. Truncated from 5’ end and about half the gene remains as a pseudogene.
Co. compressa	KU053956	25,391	74.3	—	rpl20: Truncated and here considered a pseudogene.
Corallina officinalis	KU641510	26,504	69.9		Although the manuscript is published, not yet available on GenBank Reported use of GTG (nad2) and ATT (sdhC) as start codons, otherwise reported as complete
D. binghamiae	KX247283	26,052	77.4		sdhC: Found, as unannotated 21,403 > 21,789 in conserved location between atp9 and sdhB. rpl20: No good evidence of rpl20 homology rps3: Truncated version published seems to be the result of homopolymer sequence error, though possibility of pseudogene remains. Insertion of a nucleotide around 23,590 restores conservation of start and end of gene compared with other red algal rps3 genes rps11: Moving the initiation codon from 12,194 to 12,044 removes overlap with 3’ of the nad3 gene and results in conserved start with other Florideophyceae rps11 genes based on alignment.
G. elegans	KF290995	24,922	70.5	rpl20	rpl20: With ATA start codon, gene is present but short (177 bp) with some conserved residues. Absent with ATG start. Area somewhat conserved but appears to be degraded too much to actually encode rpl20.
G. vagum	KC875854	24,901	69.5	rpl20	rpl20: With ATA start codon, gene is mostly present (222 bp) with some conserved residues. With ATG start it could be either 297 bp with overlapping tRNAs or short at 186 bp. Still considered a pseudogene here.
Gracilaria chilensis	KP728466	26,898	72.4	—	Complete
Gracilaria salicornia	KF852534	25,272	71.6	—	Complete
G. vermiculophylla	KJ526627	25,973	71.9	—	Complete
Gr. oryzoides	NC_014771 and KX687879	25,161	71.9	atp8, sdhC	atp8: Present and complete, early stop codon assumed to be sequencing error. sdhC: Present and complete, early stop codon assumed to be sequencing error. Newly sequenced Gr. oryzoides mitochondrion available at GenBank (KX687879).
G. andersonii	NC_014772 and KX687878	27,036	72	atp4 (as ymf39)	atp4: Present in resequenced G. andersonii mitochondrion. rps11: Originally reported as inversion which appears to result from frameshift mutation in original sequence. This is corrected in resequenced mitochondrion. Newly sequenced G. andersonii mitochondrion available at GenBank (KX687878).
G. chorda	NC_023251	26,534	72.4	atp4 (as ymf39)	atp4: Annotated as gene/hypothetical protein CDS in GenBank but considered as “present” in Yang et al. (2015). Definitely present.
Gracilariopsis lemaneiformis	JQ071938	25,883	72.5	—	Complete
Gra. angusta	NC_023094	27,943	69.8	—	Complete, but uses multiple start codons, seemingly where unnecessary (see table 4). Contains hypothetical protein CDS in cox1 intron.
Grateloupia taiwanensis	KM999231	28,906	68.6	—	Complete. Contains hypothetical protein CDS in cox1 intron that is also annotated as cox1. atp4: Initiation codon questionable, see table 4.
H. rubra	KF649304	33,066	67.8	atp4, atp8, rpl20	atp4: Found, as unannotated 13,225 > 13785. ORF was at conserved location immediately after cox3. Translation conserved with other Florideophyceae atp4 genes. atp8: Found, as unannotated 24,587 > 24,213. ORF present in conserved location between atp8 and nad5, 375 bp and conserved residues at n-terminus. rpl20: Truncated 5′ region–135 bp, here considered a pseudogene.
K. striatus	KF833365	25,242	69.9	atp4 (as ymf39)	atp4: Annotated as atp4 gene. This is a case of nomenclature causing confusion.
Mastocarpus papillatus	KX525587	25,906	65.0		Complete
P. palmata	KF649305	29,735	67.8	—	Complete
P. pulvinata	HQ586061	25,894	76.1	atp8, nad4L	atp8: Found, as annotated gene/hypothetical protein CDS in GenBank. 20,389 > 19,985. nad4L: Found, as unannotated 25,592 > 25,894. atp4: Moving the initiation codon from 7,772 to 7,790 will remove overlap with 3’ of the cox3 gene and result in conserved start with other Florideophyceae atp4 genes based on alignment.
Pl. cartilagineum	KJ398160	26,431	76.4	atp8, nad4L	atp8: Present, as annotated gene/ATP synthase F0 subunit 8 CDS in GenBank. 20,528 > 20,127. nad4L: Found, as unannotated 26,172 > 43 (linear sequence of circular molecule ends and starts over from the beginning of sequence). atp4: Moving the initiation codon from 7,766 to 7,784 will remove overlap with 3’ of the cox3 gene and result in conserved start with other Florideophyceae atp4 genes based on alignment.
R. pseudopalmata	KC875852	26,351	70.5	rpl20	rpl20: ATG start codon leaves it 40 residues shorter than other, while ATT start codon leaves it four residues shorter than others. Here considered present with ATT start. RNA for this gene would be very useful to confirm whether this is transcribed and not a pseudogene.
Riquetophycus sp.	KJ398161	26,351	74.3	—	Complete
S. schousboei	KJ398162	25,906	73.3	rpl20	rpl20: Found, as unannotated 23,912 > 24,148.
Sc. dubyi	KJ398163	26,438	74.1	atp4 (as ymf39), rpl20	atp4: Annotated in GenBank as “gene/hypothetical protein CDS”, BlastP of translated sequence hits other red algal atp4 and ATP synthase B chain precursor sequences and aligns well with other translated red algal atp4 genes. In Yang et al. (2015), considered atp4. rpl20: Annotated in GenBank as ‘gene/hypothetical protein CDS’, BlastP of translated sequence shows similarity to other red algal rpl20 genes.
S. flabellata	KJ398164	26,767	71.5	atp4 (as ymf39), sdhC	atp4: Annotated in GenBank as “gene/hypothetical protein CDS”, BlastP of translated sequence hits other red algal atp4 and ATP synthase B chain precursor sequences and aligns well with other translated red algal atp4 genes. In Yang et al. (2015), considered atp4. sdhC: Found a conserved ORF that relies on a TTA start codon (11,358 > 10,966).
Sp. durum	KF186230	26,202	71.6	rps11, rpl20	rpl20: Many options exist for start codons other than ATG, clearly conserved residues present. Considered present in this study using ATT start codon table 4. rps11: Pseudogene or sequencing error, regional conservation remains.
V. lanosa	KX687880	25,119	71.7	rpl20	rpl20: Likely an actual loss, all sequenced members of Ceramiales also missing rpl20. Truncated from 5′ end and about half the gene remains as a pseudogene.

Note.—Genes previously reported as missing are listed along with notes regarding their status as a result of this study.