Complete mitochondrial and chloroplast DNA sequences of the freshwater green microalga Medakamo hakoo

ABSTRACT

We report the complete organellar genome sequences of an ultrasmall green alga, Medakamo hakoo strain M-hakoo 311, which has the smallest known nuclear genome in freshwater green algae. Medakamo hakoo has 90.8-kb chloroplast and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein-coding genes, respectively. The mitochondrial genome is the smallest in the Trebouxiophyceae algae studied so far. The GC content of the nuclear genome is 73%, but those of chloroplast and mitochondrial genomes are 41% and 35%, respectively. Codon usages in the organellar genomes have a different tendency from that in the nuclear genome. The organellar genomes have unique characteristics, such as the biased encoding of mitochondrial genes on a single strand and the absence of operon structures in chloroplast ribosomal genes. Medakamo hakoo will be helpful for understanding the evolution of the organellar genome and the regulation of gene expression in chloroplasts and mitochondria.

MAIN

The sequencing of genomes across a diverse range of organisms has yielded a wealth of knowledge, shedding light not only on biodiversity but also on the existence of universal genes that are shared among all living entities. These insights will be updated as new species are discovered; therefore, the exploration of novel species is an ongoing and crucial endeavor. In particular, microalgae remain largely unexplored and are likely to contain new species that are important for evolutionary studies and industrial applications. We found an ultrasmall green alga, Medakamo hakoo strain M-hakoo 311, in a freshwater aquarium (Kuroiwa et al., 2015, 2016; Kato et al., 2023). Evolutionary analyses with ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit genes suggest that M. hakoo belongs to a new genus within the class Trebouxiophyceae (Kato et al., 2023). The nuclear genome comprises 15.8 Mbp and 7,629 genes, and is among the smallest known genomes in the Viridiplantae.

Whole-genome sequencing was performed with the PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA), and the sequence reads were assembled de novo with the RS HGAP Assemble.3 program run by SMRT Analysis software 2.3.0 (Pacific Biosciences). Genes encoded by the chloroplast/mitochondrial genomes were annotated using GeSeq employing default parameters (Tillich et al., 2017) with the following Trebouxiophyceae organellar genomes as references, since M. hakoo belongs to Trebouxiophyceae (Kato et al., 2023): Chlorella vulgaris (NC_001865/NC_045362), Coccomyxa subellipsoidea C-169 (NC_015084/NC_015316) and Botryococcus braunii (NC_025545/NC_027722). rRNA was predicted using StructRNAfinder (Arias-Carrasco et al., 2018) and RNAweasel (Lang et al., 2007). The annotations were corrected using DNADynamo (Blue Tractor Software, Llanfairfechan, UK).

We obtained two contigs representing the organellar genomes with circular sequences (Fig. 1A and 1B). The chloroplast genome (cpDNA, Fig. 1A) was 90,934 bp in length, displaying a GC content of 41.4%. We annotated 112 genes (Table 1), including 31 photosynthesis-related genes (components of photosystem I and II, the cytochrome b₆f complex, and ATP synthase subunits) and 29 tRNA and two rRNA genes. The cpDNA of M. hakoo is slightly smaller than that of its closest relative, Choricystis parasitica (Lemieux et al., 2014), which is 94,206 bp. Generally, cpDNA of green algae shows a quadripartite structure, which contains two copies of inverted repeat sequences (IRs) separated by unequal single-copy sequences, and the IRs always include the ribosomal RNA genes (Green, 2011). However, the complete cpDNA of M. hakoo lacks the quadripartite structure, similar to C. parasitica, because cpDNA of M. hakoo harbors only a single copy of the rrnL (23S ribosomal RNA) and rrnS (16S ribosomal RNA) genes (Fig. 1A). Despite most genes being shared between M. hakoo and C. parasitica, the cpDNAs of these two algae diverge regarding gene order. As expected, M. hakoo cpDNA exhibited a relatively small size and high gene density (Table 1). Excluding non-photosynthetic and parasitic algae such as Helicosporidium and Prototheca (de Koning and Keeling, 2006; Suzuki et al., 2018), the cpDNA of M. hakoo is the second smallest after Coccomyxa sp. strain SUA001 (MF805805) in Trebouxiophyceae (Table 1).

Fig. 1. Complete chloroplast (A) and mitochondrial (B) genomes of Medakamo hakoo. Both genomes were plotted using OrganellarGenomeDRAW (Greiner et al., 2019). The inner gray circle illustrates the GC content of the genome.

Table 1. Chloroplast genome sizes of known Trebouxiophyceae algae

Species	Strain	Accession	Genome size (bp)	Gene number	Coding region (%)	Note	Reference
Chlorellales
Auxenochlorella protothecoides		KC631634	84,576	109	80.29		https://www.ncbi.nlm.nih.gov/nuccore/KC631634.1
Auxenochlorella protothecoides	UTEX 2341	KY613608	84,577	106	83.68		https://www.ncbi.nlm.nih.gov/nuccore/KY613608.1
Auxenochlorella pyrenoidosa	FACHB-5	MN128434	107,442	115	66.86		https://www.ncbi.nlm.nih.gov/nuccore/MN128434.1
Chlorella heliozoae		KY629616	124,353	112	58.29		Fan et al. (2017)
Chlorella mirabilis	SAG 38.88	KM462865	167,972	115	47.64		Lemieux et al. (2014)
Chlorella sorokiniana		KJ397925	109,811	113	64.45		Orsini et al. (2016)
Chlorella sorokiniana	isolate 1230	KJ742376	109,803	111	63.07		https://www.ncbi.nlm.nih.gov/nuccore/KJ742376.1
Chlorella sp.	ArM0029B	KF554427	119,989	115	59.52		Jeong et al. (2014)
Chlorella sp.	ATCC 30562	KY629617	124,881	112	57.96		Fan et al. (2017)
Chlorella variabilis		HQ914635	124,579	115	57.14		https://www.ncbi.nlm.nih.gov/nuccore/HQ914635.1
Chlorella variabilis	DT025	MZ647689	118,106	111	59.91		https://www.ncbi.nlm.nih.gov/nuccore/MZ647689
Chlorella variabilis	NC64A	KP271969	124,793	113	58.28		Orsini et al. (2016)
Chlorella vulgaris		MT577052	165,412	114	44.64		Wen and Wan (2020)
Chlorella vulgaris		MW900257	156,202	108	42.25		https://www.ncbi.nlm.nih.gov/nuccore/MW900257.1
Chlorella vulgaris		NC_001865	150,613	210	62.39		Wakasugi et al. (1997)
Chlorella vulgaris	ITBBA3-12	MT920676	168,369	104	34.99		Han et al. (2021)
Chlorella vulgaris	NJ-7	MK948100	154,201	115	47.84		https://www.ncbi.nlm.nih.gov/nuccore/MK948100
Chlorella vulgaris	UTEX 259	MK948102	176,851	115	41.78		https://www.ncbi.nlm.nih.gov/nuccore/MK948102
Dicloster acuatus	SAG 41.98	KM462885	169,201	128	49.81		Lemieux et al. (2014)
Geminella minor	SAG 22.88	KM462883	129,187	120	61.11		Turmel et al. (2009)
Helicosporidium sp.	ex Simulium jonesii	DQ398104	37,454	54	95.18	parasitic	de Koning and Keeling (2006)
Marvania geminata	SAG 12.88	KM462888	108,470	114	64.88		Turmel et al. (2009)
Micractinium conductrix		KY629620	129,436	112	62.31		Fan et al. (2017)
Micractinium pusillum	CCAP 232/1	MN649872	115,638	105	58.15		https://www.ncbi.nlm.nih.gov/nuccore/MN649872.1
Micractinium simplicissimum		OP448647	123,552	114	58.1		https://www.ncbi.nlm.nih.gov/nuccore/OP448647
Micractinium singularis	MM0003	MN894287	139,597	106	47.85		ncbi.nlm.nih.gov/nuccore/MH983006.1
Micractinium sp.	LBA 32	MH983006	109,688	113	64.36		https://www.ncbi.nlm.nih.gov/nuccore/MH983006.1
Nannochloris desiccata	UTEX 2526	OK569791	93,001	107	73.62		Sanders et al. (2022)
Parachlorella kessleri	SAG 211-11g	FJ968741	123,994	126	64.8		Turmel et al. (2009)
Picochlorum soloecismus	DOE 101	MG552671	72,741	64	70.96		Greshake Tzovaras et al. (2020)
Picochlorum sp.	BH-2019	OQ942923	74,367	100	77.67		Dahlin et al. (2019)
Planctonema lauterbornii	SAG 68.94	KM462880	114,128	123	67.33		Lemieux et al. (2014)
Prototheca cutis		AP018373	51,673	72	92.44	parasitic	Suzuki et al. (2018)
Prototheca stagnorum		AP018372	48,188	52	84.54	parasitic	Suzuki et al. (2018)
Pseudochloris wilhelmii	SAG 1.80	KM462886	109,775	126	74.25		Lemieux et al. (2014)
Microthamniales
Fusochloris perforata	SAG 28.85	KM462882	148,459	107	50.86		Lemieux et al. (2014)
Microthamnion kuetzingianum	UTEX 318	KM462876	158,609	107	45.89		Lemieux et al. (2014)
Elliptochloris bilobata	CAUP H7103	KM462887	134,677	115	58.45		Lemieux et al. (2014)
Koliella corcontica	SAG 24.84	KM462874	117,543	128	74.24		Lemieux et al. (2014)
Koliella longiseta	UTEX 339	KM462868	197,094	118	42.48		Lemieux et al. (2014)
Prasiolopsis sp.	SAG 84.81	KM462862	306,152	115	32.81		Lemieux et al. (2014)
Stichococcus bacillaris	UTEX 176	KM462864	116,952	115	83.68		Lemieux et al. (2014)
Trebouxiales
Botryococcus braunii	SAG 807-1	KM462884	172,826	114	52.53		Lemieux et al. (2014)
Ettlia pseudoalveolaris/ Parietochloris pseudoalveolaris	UTEX 975	KM462869	126,694	125	66.18		Lemieux et al. (2014)
Lobosphaera incisa		KM821265	156,028	111	49.97		Tourasse et al. (2015)
Lobosphaera incisa	SAG 2007	KM462871	156,031	111	56.45		Lemieux et al. (2014)
Myrmecia israelensis	UTEX 1181	KM462861	146,596	112	52.59		Lemieux et al. (2014)
Pabia signiensis/ Pseudochlorella signiensis	SAG 7.90	KM462866	236,463	118	36.12		Lemieux et al. (2014)
Symbiochloris handae	SAG 2150	KM462860	289,394	116	36.08		Lemieux et al. (2014)
Symbiochloris sp.	SG-2018	ON645925	158,961	126	70.98		https://www.ncbi.nlm.nih.gov/nuccore/ON645925.1
Xylochloris irregularis	CAUP H7801	KM462872	181,542	140	57.65		Lemieux et al. (2014)
Trebouxiophyceae ordo incertae sedis
Choricystis parasitica	SAG 17.98	KM462878	94,206	111	79.12		Lemieux et al. (2014)
Coccomyxa subellipsoidea	C-169	HQ693844	175,731	115	44.94		https://www.ncbi.nlm.nih.gov/nuccore/HQ693844.1
Coccomyxa sp.	Obi	AP025008	177,965	243	66.43		https://www.ncbi.nlm.nih.gov/nuccore/AP025008.1
Coccomyxa sp.	SUA001	MF805805	68,605	100	71.18		https://www.ncbi.nlm.nih.gov/nuccore/MF805805
Leptosira terrestris/ Pleurastrum terricola	UTEX 333	EF506945	195,081	119	48.55		de Cambiaire et al. (2007)
Paradoxia multiseta	SAG 18.84	KM462879	183,394	125	51.23		Lemieux et al. (2014)
Medakamo hakoo	M-hakoo 311 (=NIES-4000)	LC604816	90,934	112	81.93		this study
Trebouxiophyceae sp.	MX-AZ01	JX402620	149,707	115	54.15		Servín-Garcidueñas and Martínez-Romero (2012)
Watanabeales
Calidiella yingdensis	D201	ON986222	132,956	108	58.53		Liu et al. (2023)
Chloroidium sp.	KL-2023a clone 2023	ON986219	239,655	113	37.65		Liu et al. (2023)
Kalinella pachyderma	clone 2601	ON986220	273,947	113	36.47		Liu et al. (2023)
Massjukichlorella minus	clone BN2720	ON986221	240,832	120	41.21		Liu et al. (2023)
Phyllosiphon coccidium	clone S213	ON986218	152,197	109	54.41		Liu et al. (2023)
Polulichloris maxima	clone L123	ON986224	165,748	111	49.66		Liu et al. (2023)
Watanabea reniformis	SAG 211-9b	KM462863	201,425	118	45.68		Lemieux et al. (2014)
Watanabea sichuanensis	H051	ON986223	208,433	118	43.83		Liu et al. (2023)

Chloroplast genome data registered as a complete genome were collected from the NCBI database, from which the genome size, the number of genes and the coding region were obtained. Counting was automatically processed based on information in each accession, so there may be slight miscounting depending on the original data.

The mitochondrial genome (mtDNA) of M. hakoo was 36,544 bp and had a GC content of 35.2% (Fig. 1B). It is the smallest of the known Trebouxiophyceae mtDNAs and exhibits a notably high gene density (85.2%, Table 2). In general, changes in organellar genome size are known to correlate with the length of intergenic regions if the organelles keep their functions (Smith and Keeling, 2015). Consistent with this, the mtDNA of M. hakoo contains relatively short intergenic regions. The mtDNA comprises 59 genes (Table 2) including 25 tRNAs, three rRNAs, 18 respiratory chain complex genes, an ATP synthase gene, a subunit C gene of twin arginine translocation (tatC) and a homing endonuclease gene (LHE). Notably, almost all the genes were predicted to be transcribed from the same strand. This characteristic is also observed in Trebouxiales, including Trebouxiophyceae sp. MX-AZ01 (Servín-Garcidueñas and Martínez-Romero, 2012), Coccomyxa subellipsoidea C-169 (Smith et al., 2011) and Botryococcus braunii (Blifernez-Klassen et al., 2016; Martínez-Alberola et al., 2019). However, the group II introns of the trnH (gug) and trnW (cca) genes, which are conserved in these species (Martínez-Alberola et al., 2019), were not predicted in M. hakoo. Typically, in Trebouxiophyceae, the rpl10 gene is located next to rps19 (Martínez-Alberola et al., 2019), but M. hakoo had another ORF in this position that was not similar to other known genes.

Table 2. Mitochondrial genome sizes of known Trebouxiophyceae algae

Species	Strain	Accession	Genome size (bp)	Gene number	Coding region (%)	Note	Reference
Chlorellales
Auxenochlorella protothecoides		KC843974	57,274	68	73.4		https://www.ncbi.nlm.nih.gov/nuccore/KC843974
Auxenochlorella protothecoides	UTEX 2341	KY681419	57,063	70	68.4		https://www.ncbi.nlm.nih.gov/nuccore/KY681419
Chlorella heliozoae		KY629615	62,477	61	63.1		Fan et al. (2017)
Chlorella ohadii		MT995630	52,560	60	61.8		https://www.ncbi.nlm.nih.gov/nuccore/MT995630
Chlorella sorokiniana	isolate 1230	KJ742377	52,528	61	64.1		https://www.ncbi.nlm.nih.gov/nuccore/KJ742377
Chlorella sp.	ArM0029B	KF554428	65,049	65	54.2		Jeong et al. (2014)
Chlorella sp.	ATCC 30562	KY629618	79,601	62	50.5		Fan et al. (2017)
Chlorella variabilis	NC64A	KP271968	78,500	62	50.0		Orsini et al. (2016)
Chlorella vulgaris	NJ-7	MK948101	87,477	65	42.6		https://www.ncbi.nlm.nih.gov/nuccore/MK948101
Chlorella vulgaris	UTEX 259	MK948103	98,062	67	40.0		https://www.ncbi.nlm.nih.gov/nuccore/1778222418
Chlorella vulgaris	ITBBA3-12	MT419367	88,754	33	30.2		Hu et al. (2020)
Chlorella vulgaris		MW900258	84,368	64	42.8		https://www.ncbi.nlm.nih.gov/nuccore/MW900258.1
Helicosporidium sp.	ex Simulium jonesi	GQ339576	49,343	66	75.7	parasitic	Pombert and Keeling (2010)
Micractinium conductrix		KY629619	74,708	62	51.6		Fan et al. (2017)
Micractinium pusillum	CCAP 232/1	MN649871	70,061	58	46.1		https://www.ncbi.nlm.nih.gov/nuccore/MN649871
Micractinium singularis	MM0003	MN894286	75,931	59	43.5		Jo et al. (2020)
Micractinium sp.	LBA 32	MH718999	77,435	63	53.6		https://www.ncbi.nlm.nih.gov/nuccore/MH718999
Micractinium variabile	KSF0031	MT332838	65,047	62	47.5		Kim et al. (2021)
Nannochloris desiccata	UTEX 2526	OK569792	40,231	69	83.6		Sanders et al. (2022)
Picochlorum soloecismus	DOE 101	MG552670	38,672	32	62.3		Gonzalez-Esquer et al. (2018)
Prototheca bovis	SAG 2021	MF197534	39,222	61	76.9	parasitic	Severgnini et al. (2018)
Prototheca ciferrii	SAG 2063	MF197533	38,164	59	73.7	parasitic	Severgnini et al. (2018)
Prototheca wickerhamii		NC_001613	55,328	63	70.6	parasitic	Wolff et al. (1994)
Trebouxiales
Botryococcus braunii	Showa	KR057902	84,583	66	54.3		Zou and Bi (2016)
Lobosphaera incisa	SAG 2468	KP902678	69,997	64	57.1		Tourasse et al. (2015)
Symbiochloris sp.	SG-2018	ON897766	59,508	58	57.8		https://www.ncbi.nlm.nih.gov/nuccore/ON897766.1
Trebouxia sp.	TR9	MH917293	70,070	63	62.1		Martínez-Alberola et al. (2019)
Trebouxia sp.	A1-2	MN642622	99,907	78	43.9		Greshake Tzovaras et al. (2020)
Trebouxiophyceae ordo incertae sedis
Coccomyxa sp.	C-169	HQ874522	65,497	60	59.1		Smith et al. (2011)
Medakamo hakoo	M-hakoo 311 (=NIES-4000)	LC604817	36,544	59	85.2		this study
Trebouxiophyceae sp.	MX-AZ01	JX315601	74,423	67	68.2		Servín-Garcidueñas and Martínez-Romero (2012)
Watanabeales
Chloroidium sp.	UTEX 3077	MN646686	90,774	54	50.7		https://www.ncbi.nlm.nih.gov/nuccore/MN646686
Jaagichlorella hainangensis		MN966687	54,084	52	58.3		https://www.ncbi.nlm.nih.gov/nuccore/MN966687
Jaagichlorella roystonensis		MN934958	130,507	64	34.0		Ma et al. (2020)

Mitochondrial genome data registered as complete genomes were obtained from the NCBI database, from which the genome size, the number of genes and the coding region were obtained.

We then analyzed the codon usage of both chloroplasts and mitochondria (Table 3). There were 25 codons in chloroplasts and 27 in mitochondria with a relative synonymous codon usage (RSCU) exceeding 1, i.e., codons with higher usage frequencies than the theoretical expectation. All these codons in mitochondria and 23 out of 25 in chloroplasts ended in A or T nucleotides. The coding sequences of the cpDNA and mtDNA exhibited GC contents of 44.2% and 37.7%, respectively, closely matching the GC contents of their entire organellar genomes. However, the GC contents of the 3rd position (31.6% and 21.0% for cpDNA and mtDNA, respectively) were markedly lower, which is a unique characteristic of the organellar genomes of M. hakoo. We have already reported that the GC content of the nuclear genome of M. hakoo is exceptionally high, at 73%, and it has been pointed out that amino acids corresponding to codons with higher GC usage tend to be preferentially employed in protein synthesis (Kato et al., 2023). On the other hand, the alteration in amino acid composition due to such a bias could not be observed in the organelle genome. In addition, neither mitochondria nor chloroplasts necessarily have a high codon usage rate corresponding to tRNA encoded within their genomes.

Table 3. Codon usage analysis of chloroplast and mitochondrial genomes in Medakamo hakoo

Chloroplast genome
Triplet	AA	Fraction	FPT	Number	RSCU	Triplet	AA	Fraction	FPT	Number	RSCU	Triplet	AA	Fraction	FPT	Number	RSCU	Triplet	AA	Fraction	FPT	Number	RSCU
TTT	F	0.61	33.0	738	1.21	TCT	S	0.24	14.3	320	1.44	TAT	Y	0.56	17.7	396	1.13	TGT	C	0.65	6.3	140	1.29
TTC	F	0.39	21.5	480	0.79	TCC	S	0.04	2.6	58	0.26	TAC	Y	0.44	13.6	305	0.87	TGC	C	0.35	3.4	77	0.71
TTA	L	0.37	38.3	856	2.23	TCA	S	0.29	17.0	381	1.71	TAA	*	0.81	2.9	64	–	TGA	*	0.04	0.1	3	–
TTG	L	0.10	10.0	223	0.58	TCG	S	0.15	9.0	201	0.90	TAG	*	0.15	0.5	12	–	TGG	W	1.00	14.8	331	1.00
CTT	L	0.31	31.8	712	1.85	CCT	P	0.31	15.8	354	1.24	CAT	H	0.46	9.6	215	0.92	CGT	R	0.59	36.3	811	3.51
CTC	L	0.07	7.0	157	0.41	CCC	P	0.08	3.8	86	0.30	CAC	H	0.54	11.3	253	1.08	CGC	R	0.14	8.7	194	0.84
CTA	L	0.09	9.1	204	0.53	CCA	P	0.45	23.1	516	1.81	CAA	Q	0.83	37.2	832	1.66	CGA	R	0.17	10.4	233	1.01
CTG	L	0.07	6.9	154	0.40	CCG	P	0.16	8.2	183	0.64	CAG	Q	0.17	7.7	173	0.34	CGG	R	0.04	2.5	55	0.24
ATT	I	0.69	41.8	936	2.06	ACT	T	0.31	18.2	408	1.25	AAT	N	0.48	17.5	392	0.96	AGT	S	0.20	11.8	264	1.19
ATC	I	0.28	17.2	384	0.84	ACC	T	0.10	5.7	127	0.39	AAC	N	0.52	19.1	427	1.04	AGC	S	0.08	5.0	111	0.50
ATA	I	0.03	2.0	45	0.10	ACA	T	0.40	23.2	518	1.59	AAA	K	0.80	41.7	933	1.60	AGA	R	0.05	3.0	68	0.29
ATG	M	1.00	24.1	540	1.00	ACG	T	0.19	11.1	249	0.76	AAG	K	0.20	10.3	230	0.40	AGG	R	0.02	1.1	25	0.11
GTT	V	0.47	32.7	732	1.90	GCT	A	0.40	31.3	701	1.60	GAT	D	0.66	28.5	638	1.32	GGT	G	0.57	40.0	895	2.29
GTC	V	0.13	8.6	193	0.50	GCC	A	0.13	10.4	233	0.53	GAC	D	0.34	14.7	329	0.68	GGC	G	0.13	9.4	211	0.54
GTA	V	0.24	16.6	372	0.96	GCA	A	0.28	21.9	490	1.12	GAA	E	0.75	39.6	885	1.51	GGA	G	0.16	11.4	255	0.65
GTG	V	0.16	11.0	246	0.64	GCG	A	0.19	14.6	326	0.75	GAG	E	0.25	12.9	288	0.49	GGG	G	0.13	9.1	203	0.52

Mitochondrial genome
Triplet	AA	Fraction	FPT	Number	RSCU	Triplet	AA	Fraction	FPT	Number	RSCU	Triplet	AA	Fraction	FPT	Number	RSCU	Triplet	AA	Fraction	FPT	Number	RSCU
TTT	F	0.70	49.5	420	1.39	TCT	S	0.28	19.9	169	1.67	TAT	Y	0.69	26.4	224	1.39	TGT	C	0.82	10.6	90	1.64
TTC	F	0.30	21.7	184	0.61	TCC	S	0.02	1.7	14	0.14	TAC	Y	0.31	11.7	99	0.61	TGC	C	0.18	2.4	20	0.36
TTA	L	0.55	68.9	584	3.33	TCA	S	0.31	22.2	188	1.86	TAA	*	0.94	3.7	31	–	TGA	*	0.06	0.2	2	–
TTG	L	0.04	5.0	42	0.24	TCG	S	0.08	5.5	47	0.46	TAG	*	0.00	0.0	0	–	TGG	W	1.00	19.6	166	1.00
CTT	L	0.27	33.5	284	1.62	CCT	P	0.38	15.9	135	1.51	CAT	H	0.56	11.8	100	1.11	CGT	R	0.48	21.9	186	2.89
CTC	L	0.01	1.5	13	0.07	CCC	P	0.02	0.9	8	0.09	CAC	H	0.44	9.4	80	0.89	CGC	R	0.10	4.4	37	0.58
CTA	L	0.09	11.6	98	0.56	CCA	P	0.49	20.6	175	1.96	CAA	Q	0.89	34.6	293	1.77	CGA	R	0.25	11.6	98	1.52
CTG	L	0.03	3.8	32	0.18	CCG	P	0.11	4.6	39	0.44	CAG	Q	0.11	4.5	38	0.23	CGG	R	0.01	0.4	3	0.05
ATT	I	0.73	53.4	453	2.20	ACT	T	0.42	22.9	194	1.68	AAT	N	0.66	22.5	191	1.32	AGT	S	0.26	18.9	160	1.58
ATC	I	0.16	11.8	100	0.48	ACC	T	0.05	2.7	23	0.20	AAC	N	0.34	11.6	98	0.68	AGC	S	0.05	3.4	29	0.29
ATA	I	0.11	7.8	66	0.32	ACA	T	0.44	23.9	203	1.76	AAA	K	0.78	40.0	339	1.57	AGA	R	0.15	6.8	58	0.90
ATG	M	1.00	25.8	219	1.00	ACG	T	0.09	4.8	41	0.36	AAG	K	0.22	11.0	93	0.43	AGG	R	0.01	0.5	4	0.06
GTT	V	0.55	39.3	333	2.21	GCT	A	0.48	33.5	284	1.94	GAT	D	0.78	24.7	209	1.57	GGT	G	0.30	18.5	157	1.18
GTC	V	0.02	1.8	15	0.10	GCC	A	0.06	4.1	35	0.24	GAC	D	0.22	6.8	58	0.43	GGC	G	0.02	1.1	9	0.07
GTA	V	0.33	23.8	202	1.34	GCA	A	0.38	26.1	221	1.51	GAA	E	0.78	29.4	249	1.56	GGA	G	0.57	35.5	301	2.27
GTG	V	0.09	6.3	53	0.35	GCG	A	0.08	5.5	47	0.32	GAG	E	0.22	8.4	71	0.44	GGG	G	0.12	7.5	64	0.48

If a transfer RNA (tRNA) is encoded within the organelle’s DNA, the Triplet column entry is colored yellow. Additionally, entries in the RSCU (relative synonymous codon usage) column are colored when the value exceeds 1. AA: amino acid in one-letter code; FPT: frequency per thousand.

As mentioned above, most genes are encoded unilaterally in the mtDNA in M. hakoo. This feature has been confirmed in closely related species, including Coccomyxa sp. and Trebouxiophyceae sp. MX-AZ01, but also in B. braunii and Symbiochloris sp. (ON897766) in Trebouxiales, and Jaagichlorella in Watanabeales. Many species in this Choricystis/Botryococcus clade share the characteristic that the ribosomal genes are dispersed in the cpDNA and are unlikely to form an operon. It will be necessary for future algal research to focus on how mitochondrial genes transcribed from a single strand are expressed (whether they create operons) and, conversely, how the separately encoded chloroplast rRNA genes, which typically form an operon structure, exhibit coordinated transcription.

PUBLIC AVAILABILITY AND ACCESSION NUMBERS

Medakamo hakoo is stored as strain NIES-4000 in the Microbial Culture Collection at the National Institute for Environmental Studies (NIES Collection, Tsukuba, Japan). The cpDNA and mtDNA genome sequences were deposited in DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/index-e.html) with accession numbers LC604816 for chloroplasts and LC604817 for mitochondria. The associated BioProject, SRA and BioSample accession numbers are PRJNA771468, SRR16480670, SRR16480671, SRR16480672, SRR16480673 and SAMN22310827, respectively.

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16H04813 and 19H03260) to T. K. and by JST-CREST (JPMJCR20S6), JST-OPERA (JPMJOP1832) and JST-GteX (JPMJGX23B0) to S. Matsunaga. The authors thank Mr. Brody Frink (Kyoto University) for editing a draft of this manuscript.

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