Background

Cryptophytes are an ecologically important group of algae comprised of phototrophic, heterotrophic and osmotrophic species. This lineage is of great interest to evolutionary biologists because their plastids are of red algal secondary endosymbiotic origin. Cryptophytes have a clear phylogenetic affinity to heterotrophic eukaryotes and possess four genomes: host-derived nuclear and mitochondrial genomes, and plastid and nucleomorph genomes of endosymbiotic origin.

Results

To gain insight into cryptophyte mitochondrial genome evolution, we sequenced the mitochondrial DNAs of five species and performed a comparative analysis of seven genomes from the following cryptophyte genera:Chroomonas,Cryptomonas,Hemiselmis,Proteomonas,Rhodomonas,Storeatula andTeleaulax. The mitochondrial genomes were similar in terms of their general architecture, gene content and presence of a large repeat region. However, gene order was poorly conserved. Characteristic features of cryptophyte mtDNAs included large syntenic clusters resembling α-proteobacterial operons that encode bacteria-like rRNAs, tRNAs, and ribosomal protein genes. The cryptophyte mitochondrial genomes retain almost all genes found in many other eukaryotes including thenad,sdh,cox,cob, andatp genes, with the exception ofsdh2 andatp3. In addition, gene cluster analysis showed that cryptophytes possess a gene order closely resembling the jakobid flagellatesJakoba andReclinomonas. Interestingly, thecox1 gene ofR. salina,T. amphioxeia, andStoreatula species was found to contain group II introns encoding a reverse transcriptase protein, as did thecob gene ofStoreatula species CCMP1868.

Conclusions

These newly sequenced genomes increase the breadth of data available from algae and will aid in the identification of general trends in mitochondrial genome evolution. While most of the genomes were highly conserved, extensive gene arrangements have shuffled gene order, perhaps due to genome rearrangements associated with hairpin-containing mobile genetic elements, tRNAs with palindromic sequences, and tandem repeat sequences. Thecox1 andcob gene sequences suggest that introns have recently been acquired during cryptophyte evolution. Comparison of phylogenetic trees based on plastid and mitochondrial genome data sets underscore the different evolutionary histories of the host and endosymbiont components of present-day cryptophytes.

Electronic supplementary material

The online version of this article (10.1186/s12864-018-4626-9) contains supplementary material, which is available to authorized users.

Keywords: Cryptophytes, Genome re-arrangement, Mitochondrial genome, Mobile genetic elements

General features of cryptophyte mitochondrial genomes

Mitochondrial genomes (mtDNAs) were sequenced and analyzed from representatives of three different colored cryptophytes: the red-colouredProteomonas,Rhodomonas,Storeatula andTeleaulax, the green-colouredChroomonas andHemiselmis, and the brown-coloredCryptomonas species (Table 1). The mitochondrial genomes of two species,Storeatula sp. CCMP1868 andTeleaulax amphioxeia, could not be completely assembled, due to the presence of complex repeat regions (Fig. 1), as seen previously (e.g., in the haptophytePhaeocystis; [23]). All of the newly sequenced cryptophyte mitochondrial genomes were found to have a highly repetitive non-coding region (Table1), as previously described in two other cryptophyte mitochondrial genomes [20,21]. The mitochondrial genomes ranged in size from ~ 37 Kbp (Cryptomonas curvata) to ~ 54.5 Kbp (Storeatula sp. CCMP 1868), not including repeat regions (Table1). They share a core set of 2 rRNAs, 25~ 28 tRNAs and 42 protein-coding genes, including 18 components of the respiratory chain, 5 ATP synthase subunits, 16 ribosomal proteins and 2 subunits of thetat translocase (Tables 2,3). Notably, the mitochondrion-encodedrps1 andtatA genes in cryptophytes were previously found to be present exclusively in jakobids and/or malawimonads, believed to possess the most ‘primitive’ mitochondrial genomes known [18]. Minor variation was found in tRNA gene content (Table 2). For instance,trnK(UUU) is present in three red-colored cryptophytes,R. salina,Storeatula sp. CCMP1868, andT. amphioxeia, but notProteomonas sulcata, while the blue/green-coloredCh. placoidea andH. andersenii and the brown-coloredCr. curvata have a unique isotypetrnG(GCC) (Table2).

Further consideration of mitochondrial genome structure revealed that while all genes were located on the same strand in theChroomonas placoidea,Hemiselmis andersenii andProteomonas sulcata genomes, some genes were located on the opposite strand inCryptomonas curvata (31 genes),Storeatula sp. CCMP1868 (41 genes),Teleaulax amphioxeia (39 genes), andRhodomonas salina (53 genes) (Figs. 1,2, Table1).

Genome re-arrangements

The most evident feature of the cryptophyte mitochondrial genomes sequenced herein was that, although gene content is very stable, gene order is highly variable. The 42 protein-coding genes were arranged together and analyzed in the context of 12 syntenic blocks including rRNA and tRNA genes, each block consisting of 2–6 genes (Fig. 2). These blocks were as follows: A)cox1-cob-nad11, B)atp8-atp4-rps4-atp9, C)nad3-rps2, D)tatC-nad7, E)cox2-tatA-cox3-rpl12-rpl7-rps19, F)rps3-rpl16-rpl14-rpl5-rps14, G)nad8-nad6-nad1, H)sdh3-sdh4, I)nad4L-nad5-nad4-nad2, J)atp1-rps1-orf166, rpl31, K)rps8-rpl6-rps13-rps11-atp6, L)nad10-nad9, and 2 rRNAs (Fig.2). Two syntenic blocks were found to be common to all cryptophyte mitochondrial genomes (Fig.2, J and L).

To assess the extent of mitochondrial genome rearrangements more closely, genomes were aligned using the Mauve genome aligner (Additional file 1: Figure S1). Synteny was broadly conserved within the three differently colored cryptophyte lineages. Overall, we documented 23 instances of mitochondrial genome rearrangements, suggesting that extensive scrambling has occurred since the evolutionary split of these species (Fig.2 and Additional file1: Figure S1). ComparingProteomonas sulcata with the three other red-colored cryptophyte mitochondrial genomes, 14 gene-order rearrangements were detected (Fig.2 ①–⑭). The genes in the mitochondrial genome ofTeleaulax amphioxeia appear to have been rearranged via transposition of synteny block C (①), an inversion of a set of consecutive genes in blocks G to L (②), and a combination of transposition and inversion involving synteny block H (③). Genes in the mitochondrial genome ofRhodomonas salina were rearranged with transposition ofnad3 andrps2 from broken block C (in a manner different than that seen forT. amphioxeia) (④, ⑤), transposition of block D–E (⑥), an inversion of a set of consecutive genes in blocks G to J (⑦), and transposition ofatp6 within block K (⑧). The mitochondrial genome ofStoreatula sp. CCMP1868 was rearranged with transposition of synteny block G–L (⑨), transposition oftatC andnad7 from a broken block D (⑩), an inversion of block E (⑪), an inversion of block F (⑫), inversion of block B with rRNAs andnad11 being split from block A (⑬), and an inversion of block C (⑭).

In terms of mitochondrial gene order, the brown-colored cryptophyteCryptomonas curvata is more similar to the red-colored lineage than to the blue-greens. Relative to the genome of the red-coloredProteomonas sulcata, four gene-order rearrangements were detected (Fig.2 ⑮–⑱): a combination of an inversion and break in block E–F (⑮–⑯), an inversion of a string of genes in blocks G to L (⑰), and a combination of inversion and transposition block C (⑱). The gene order of blue/green-colored cryptophytes is very different from the other groups. Between the two blue/green-colored cryptophytesHemiselmis andersenii andChroomonas placoidea, five gene-order rearrangements were detected (Fig.2 ⑲–): transposition of G and partial I block (⑲), transposition ofrps4 (⑳) andatp9 () from broken block B, transposition ofsdh4 from broken block H (), and transposition ofcox2 break free from the block E (). Clearly there have been extensive mitochondrial genome rearrangements in each of these lineages during cryptophyte evolution.

Palindromic sequences and large non-coding regions with tandem repeats

We found between 3 and 20 A-T rich palindromic sequences in the intergenic regions of each of the mitochondrial genomes of cryptophytes (Fig. 3, Additional files 2 and3: Figures S2 and S3), similar to those reported previously in the large non-coding regions ofRhodomonas salina [20] andHemiselmis andersenii [21]. Interestingly, between 4 and 9 tRNA genes are present at the palindromic endpoints (Additional file 3: Figure S3).

Cryptophyte mtDNA structure also appears to have been impacted by the presence of tandem repeats in the large non-coding region, with individual repeats ranging from 1.1Kbp (P. sulcata) to 19.7Kbp (H. andersenii), each of which contains multi-copy palindromic sequences [20,21] (Figs.1 and2 marked as triangle). The repeats are inverted in the genomes ofR. salina,Storeatula species CCMP1186 andT. amphioxeia, but in the other four completely assembled genomes, large non-coding regions are instead dispersed or arranged in tandem throughout the large non-coding region (Table1).

Ribosomal proteins and bacterial-like operons

Ribosomal protein gene clusters have been suggested to represent vestiges of bacterial operons in the cryptophyte mitochondrial genome [20]. Our comparisons of mitochondrial gene data provide further support for this idea. The ribosomal protein clustersrps3-rpl16-rpl14-rpl5-rps14 andrps8- rpl6-rps13-rps11 inCh. placoidea,H. andersenii,Cr. curvata,R. salina andP. sulcata exhibit the same relative gene order as in the operons of the bacteriaEscherichia coli andRickettsia prowazekii; this arrangement is also seen (with little variation) in the gene-rich mtDNAs of several other protists (Jakoba libera, Acanthamoeba catellanii,Phytophthora infestans, and the green algaNephroselmis olivacea) as well as the liverwortM. polymorpha (Additional file 4: Figure S4). However, the bacterial-operon clusterrps8- rpl6-rps13-rps11 was inverted in the cryptophytesStoreatula sp. CCMP 1868 andT. amphioxeia (Additional file4: Figure S4). Furthermore, the other conserved ribosomal protein gene cluster,rps12-rps7-rps19, is translocated in the mitochondrial genome ofStoreatula sp. CCMP1868.

Phylogeny

An ML tree was reconstructed using 4257 amino acids from 16 representative genes conserved in the mtDNAs of all chlorophyll-c containing algal groups (Fig. 4), as well as diverse photosynthetic and non-photosynthetic lineages from across the eukaryotic tree. The sequences of dinoflagellates and euglenoids were not included in this analysis due to the limited available data of mitochondrial genes. A monophyletic cryptophyte clade was strongly supported (MLB = 100%) and showed well-resolved internal relationships. The four red-colored taxa (i.e.,Storeatula,Rhodomonas,Teleaulax andGuillardia) group together while the brown-coloredCryptomonas curvata forms a sister relationship with the blue/green-coloredChroomonas andHemiselmis species (Fig.4). Although our taxon sampling was limited, these results are consistent with earlier studies. In particular, the red-colored cryptophyte species (Rhodomonas,Storeatula,Teleaulax, andProteomonas) form a monophyletic lineage in both mitochondrial and plastid phylogenomic studies to the exclusion of other cryptophytes ([24], this study), in contrast to single gene analyses of nuclear SSU rDNA [6,8].

Palindromic sequences and repeat structures relative to recombination sites

The fact that palindromic sequences are found concentrated near the ends of syntenic blocks in cryptophyte mitochondrial genomes suggests that they have played a role in mediating genomic rearrangements. In fungal mtDNAs, double-hairpin elements have been suggested to act as mobile DNA elements and to mediate lateral gene transfer [34,35]. In any case, a notable consequence of the presence of such repeats is that they facilitate genome rearrangements [36–39]. A number of possible mechanisms have been proposed. Inversions may occur in a specific location due to the presence of short repeat elements subject to homologous recombination [40,41]. Even in un-rearranged plastid genomes, small inversions regularly occur in intergenic areas, caused by short inverted repeats forming hairpins that can easily flip the orientation of the intervening sequences [42,43]. Such a mechanism could explain the highly shuffled genes seen in the cryptophytes mitochondrial genomes described herein (Fig.2). In addition, tRNA genes associated with gene re-arrangement breakpoints have been reported in plastid genomes [41,44]. It has been suggested that tRNA genes at inversion endpoints may be due to the presence of short repeats within or near the tRNA genes in the highly rearranged chloroplast genome of the charophyteChaetospheridium globosum [45], as well as in the flowering plantTrachelium [46].

The direct repeat arrangements we observed are strikingly similar to the larger (35 kb) repeat structure found in the mitochondrial genome of the diatomPhaeodactylum tricornutum [47]. The haptophytesChrysochromulina tobin,Emiliania hyxleyi,Phaeocystis antarctica,Phaeocystis globosa andPavlova lutheri, and the chlorophytesPedinomonas minor andAcutodesmus obliquus also contain large tandem repeat regions (>4 kb) in their genomes [23,25,27,28,48]. In animal mitochondrial genomes, the tandem repeats are located in the control region and help explain the evolutionary origins of tandem repeats among species, populations and even individuals [49,50]. However, in most algal mitochondrial genomes the tandem repeat manifests itself in a pattern that is species-specific. The repeat sequences within the mitochondrial genomes of cryptophytes described herein do not retain obvious sequence or structural similarity across species bounds.

Gene content of cryptophyte mitochondrial genomes

The most gene-rich mitochondrial genomes reported to date are those of the excavate group Jakobida, with 61–69 protein-coding genes and 30–34 RNAs [18]. Cryptophyte mitochondrial genomes are also noteworthy in retaining genes that are not found in most other eukaryotes (Fig. 5). The mtDNAs of cryptophytes contain 42 conserved protein genes (excluding non-conserved ORFs in each genome), more than found in glaucophytes (30 CDSs), Bacillariophyceae (34 CDSs), Eustigmatophyceae (36 CDSs), Phaeophyceae (34 CDSs), Rhaphidophyceae (34 CDSs), rhodophytes (22 CDSs), haptophytes (22 CDSs), Synurophyceae (32 CDSs), chlorarachniophytes (26 CDSs) and green algae (11–39 CDSs). Most of the cryptophyte mitochondrial genes are associated with electron transport systems belonging to a set of five complexes, as summarized below.

The genes encoding certain subunits of NADH dehydrogenase (complex I;nad7,nad8,nad9,nad10, andnad11) are only rarely found in mtDNA. Thenad7,nad9 andnad11 genes are missing from all haptophyte and rhodophyte mitochondrial genomes. On the other hand, these three genes are present in all cryptophytes,Palpitomonas, stramenopiles and jakobid mitochondrial genomes (Fig.5). Comparison of the mtDNA gene order in red-/brown-colored cryptophytes reveals the presence of three NADH dehydrogenase gene clusters (cluster G:nad8-nad6-nad1, cluster I:nad4L-nad5-nad4-nad2, and cluster L:nad10-nad9). However, two clusters (G and I) were separated into one or two genes, respectively (G:nad8-nad1 andnad6, cluster I:nad4L-nad5 andnad4-nad2), in blue/green-colored cryptophytes (Fig.2).

The succinate dehydrogenase (complex II) is made up of four protein subunits; two are hydrophilic and belong to the catalytic portion of the complex (sdh1 andsdh2 genes) and the other two, encoded by thesdh3 andsdh4 genes, are hydrophobic and act as anchors to the entire complex [51,52]. Thesdh2,sdh3, andsdh4 genes are sometimes found in mitochondrial genomes, whereassdh1 is transferred to the nuclear genome and its protein product is imported from the cytosol. Thesdh2 gene is present in Rhodophyta except inGaldieria sulphuraria [31]. Many other algae have also lost thesdh2 gene from their mitochondrial genomes. Mitochondrial genes for two subunits of succinate dehydrogenase (sdh3 andsdh4) are present in only a few green algae, the liverwortMarchantia polymorpha, several red algae [31,53], all jakobid flagellates includingReclinomonas americana [54,55] and all known cryptophytes ([20,21], this study).

The genes for subunits of the cytochromebc₁ complex (complex III) and cytochromec oxidase (complex IV) are present in the mtDNAs of all cryptophytes and most algae. Thecob andcox1 genes are always together in all cryptophyte genomes sequenced thus far (Fig.2A). Thecox2 andcox3 genes are found together in almost all cryptophytes, the exception being thecox2 gene inHemiselmis andersenii, which is separated from cluster E (Fig.2E). Interestingly, thecox1 gene ofStoreatula sp. CCMP1868,Teleaulax amphioxeia, andRhodomonas salina was found to contain a putative group II intron with a coding region for an intron encoded protein (IEP). InChroomonas placoidea, thecox1 gene was split into two parts with 13 genes in between. The first exon encodes a maturase sequence and is located between thenad8-trnD andatp8 genes. Thecob gene inStoreatula sp. CCMP1868 also has an intron with an IEP (Additional file 5: Figure S5). These introns belong to group II, which often harbor three distinct protein-coding regions corresponding to reverse transcriptase, maturase, and C-terminal DNA binding domains [56]. TheStoreatula sp. intron coding regions show similarity to an IEP in theatpA gene in the mitochondrial genome of the liverwortMarchantia polymorpha [57].

The mtDNAs of jakobid flagellates contain the biggest complement of ATP synthase genes (atp1, 3, 4, 6, 8, 9). Threeatp genes (atp6, 8, 9) are present in the mitochondrial genomes of green algae, glaucophytes, haptophytes and stramenopiles (Fig.5). Theatp1 andatp4 genes are present in the mitochondrial genome of green algae, jakobids and cryptophytes. The heteroloboseanNaegleria fowleri andTsukubamonas globose (a member of the Tsukubamonadidae) haveatp3 in their mtDNA but lackatp4. All cryptophytes andPalpitomonas bilix possess most of theatp gene set (atp1, 4, 6, 8, 9) with the exception ofatp3 ([22], this study).

The two genes for subunits of the twin-arginine protein translocation system transporters (encoded by thetatA andtatC genes) are generally conserved in diverse prokaryotes as well as in plastids [58]. ThetatC homologs are present in some mitochondrial genomes [59], and the mitochondrialtatA gene was thought to be limited to the jakobids [60]. However, a recent review of algal mitochondrial genomes [61] noted the presence oftatA in some eukaryotic groups, including diatoms, raphidophytes, chrysophytes and cryptophytes; these genes are found in our newly sequenced cryptophyte genome as well (Fig.5).

Phylogenetic relationships

Curiously, our 16-protein mitochondrial genome phylogeny shows a strong monophyletic relationship between cryptophytes and rhodophytes. This is unexpected given that the cryptophyte mitochondrion is derived from the host component of the original secondary endosymbiotic partnership that gave rise to modern-day cryptophytes. Based on the monophyletic relationship between chlorophyll-c containing groups and red algae seen in many (but not all) plastid genome phylogenies, the hypothesis that a single secondary endosymbiotic uptake of a red alga in a common ancestor of all ‘chromist’ algae has been explored [24,62–70]. However, recent large-scale studies using nuclear genome data show topologies that are incongruent with this hypothesis. The occurrence of multiple secondary and serial endosymbioses has been proposed to reconcile the apparent incongruence between host and endosymbiont-associated phylogenies (e.g., [12,69,71–74]). We examined phylogenies inferred from each of the single genes/proteins used in our multi-gene tree, and could see no consistent signal for a relationship between cryptophytes and rhodophytes to the exclusion of other eukaryotic groups (Additional file 6: Figure S6). In only one of our protein trees (cob) did the cryptophytes branch withPalpitomonas bilix, to which it is clearly related on the basis of nuclear multi-gene trees (e.g., [10,12]). Clearly more mitochondrial genome data are needed from plastid-lacking lineages that are related to cryptophytes, such as goniomonads and kathablepharids. Nevertheless, our mitochondrial genome-based phylogeny is consistent with previously published phylogenies, including a 250 nuclear gene dataset [12], in suggesting that the cryptophytes and haptophytes are not specifically related to one other. The monophyly of haptophytes + Opisthokonta/Amoebozoa + Stramenopiles/Rizaria + other relatives is supported by nuclear and mitochondrial analyses ([12], this study).

DNA isolation and sequencing

Cultures ofChroomonas placoidea CCAP 978/8 andProteomonas sulcata CCMP 705 were obtained from the Culture Collection of Algae and Protozoa (CCAP), whereasStoreatula species CCMP 1868 came from the National Center for Marine Algae and Microbiota (NCMA).Teleaulax amphioxeia collected from Gomso Bay, Korea (35° 40′ N, 126° 40′ E) was established as clonal cultures from single-cell isolates and the strain is available from the Culture collection at the Chungnam National University, Korea.Cryptomonas curvata collected from Cheongyang pond, Korea (36° 30′ N, 126° 47′ E) was established as clonal cultures from single-cell isolates and the strainCryptomonas curvata FBCC 300012D is available from the Freshwater Bioresources Culture Collection at the Nakdong-gang National Institute of Biological Resources Korea. All cultures were grown in AF-6 medium [75] with distilled water for the freshwater strain (Cr. curvata) or distilled seawater for marine strains, and were maintained at 20 °C under conditions of a 14:10 light:dark cycle with 30 μmol photons·m^− 2·s^− 1 from cool white fluorescent tubes. DNA was extracted using the QIAGEN DNEasy Blood Mini Kit (QIAGEN, Valencia, CA, USA) as per manufacturer’s instructions. Next-generation sequencing (NGS) was carried out using the Ion Torrent PGM platform (Thermo Fisher Scientific, San Francisco, California, USA). Sequencing libraries were prepared using the Ion Xpress™ Plus gDNA Fragment Library Preparation Kit for 200 bp or 400 bp-sized sequencing library prearation and the Ion OneTouch™ 200 or 400 Template Kit (Thermo Fisher Scientific, San Francisco, CA, USA) according to the manufacturer’s protocol. Genomes were sequenced on an Ion Torrent Personal Genome Machine (PGM) using the Ion PGM sequencing 200 or 400 Kit (Thermo Fisher Scientific, San Francisco, CA, USA). On the MiSeq (Illumina, San Diego CA), the amplified DNA was fragmented and tagged using the NexteraXT protocol (Illumina), indexed, size selected, and pooled for sequencing using the small amplicon targeted resequencing run, which performs paired end 2 × 300 bp sequencing reads using the MiSeq Reagent Kit v3 (Illumina), according to the manufacturer’s recommendations.

Genome assembly and annotation

The raw reads obtained from both NGS platforms (i.e., Ion Torrent and Illumina MiSeq) were trimmed separately using the following settings: base = 80 bp, error threshold = 0.05, n ambiguities = 2. Assemblies were also carried out separately. For the Ion Torrent data, the assembly was carried out using MIRA4 (http://mira-assembler.sourceforge.net/docs/DefinitiveGuideToMIRA.html), whereas SPAdes 3.10 (http://bioinf.spbau.ru/spades) was used for Illumina data. For each genome, the two assemblies were compared and the most ‘complete’ assembly was chosen. The mitochondrial origin of the assembled contigs was verified according to the following criteria and Jung et al. [76]: (i) reads were mapped onto the final consensus contig for each cryptophyte mitochondrial genome using Bowtie2 (similarity = 95%, length fraction = 75%;http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) with preset options (sensitive-local: -D 15 -R 2 -N 0 -L 20 -i s,1,0.75); (ii) BLAST searches using commonly known mitochondrial genes against the entire assembly produced hits to these contigs; and (iii) genome sizes consistent with those of other photosynthetic cryptophyte mitochondrial genomes were obtained.

Protein coding genes as well as rRNA and tRNA genes were compiled from all previously sequenced cryptophyte mitochondrial genomes. Preliminary annotation of protein coding genes was performed using GeneMarkS (http://opal.biology.gatech.edu/GeneMark/). The final annotation file was checked in Geneious Pro 10.2.2 (http://www.geneious.com/) using the ORFfinder with genetic code 4 (Protozoan Mitochondrial Code). Predicted ORFs were checked manually and annotated accordingly.

Transfer RNA genes were identified using the tRNAscan-SE version 2.0 server (http://lowelab.ucsc.edu/tRNAscan-SE/) with default settings and the “Mold/Protozoan Mito” model. The rRNA genes were identified by BLASTn searches against a set of known, previously published mitochondrial rRNA sequences of cryptophytes. To determine intron types, mitochondrial genomes were submitted to the RNAweasel server (http://megasun.bch.umontreal.ca/cgi-bin/RNAweasel/RNAweaselInterface.pl). Physical maps were designed with the OrganellarGenomeDRAW program (http://ogdraw.mpimp-golm.mpg.de/).

Palindromic sequence elements were detected using the EMBOSS explorer (http://emboss.bioinformatics.nl/cgi-bin/emboss/palindrome) with the following parameters: 8 for minimum length of palindromes, 100 for maximum length of palindromes, 10 for maximum gap between elements, and no mismatches within the palindrome. To find palindromic sequences in tRNAs, the maximum gap between elements was set at 100 bp (due to the normal tRNA gene length of less 90 bp). Some detected sequences were manually excluded if a loop region was longer than a stem region. Genome sequences were deposited into the NCBI GenBank database under the following accession numbers (Table1).

Gene arrangement comparisons

Two previously published cryptophyte mitochondrial genome sequences were downloaded from GenBank [20,21]. For structural and synteny comparisons, the genomes were aligned using the Mauve Genome Alignment tool version 2.2.0 [77] with default settings. For the purposes of visualization, we arbitrarily designated the beginning of thecox1 gene as position 1 in each genome (pointing in the direction ofcob).

Phylogenetic analysis

Phylogenetic trees were constructed from datasets created by combining amino acid sequences corresponding to 16 protein coding genes from 131 mitochondrial genomes, including those of seven cryptophytes, five haptophytes, 38 stramenopiles, and 36 red algae (Additional file 7: Table S1). The 16 genes are as follows:atp6,atp8,atp9,cob,cox1,cox2,cox3,nad1,nad2,nad3,nad4,nad4L,nad5,nad6,rps12, andrpl16. The dataset was concatenated (4257 amino acid positions) and final refinements were performed by eyes in the MacGDE2.5 program [78].

Maximum likelihood (ML) phylogenetic analyses were performed using RAxML version 8.0.0 [79] with the Le and Gascuel gamma (LG + GAMMA) model [80] for amino acid data chosen by ProtTest 3 [81]. We used 1000 independent tree inferences using the -# option to identify the best tree. The model parameters with gamma correction values and the proportion of invariable sites in the combined dataset were obtained automatically by the RAxML program. Bootstrap support values (MLBS) were calculated using 1000 replicates with the same substitution model.

Funding

This research was supported by the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT & Future Planning, Basic Science Research Program (MSIP; NRF-2013R1A1A3012539) and the Ministry of Education (2015R1D1A1A01057899) to JIK; NRF (2017R1A2B3001923) and the Collaborative Genome Program (20140428) funded by the Ministry of Oceans and Fisheries, Korea to HSY; NRF (2016R1D1A1A09919318), and the Dongguk University Research Fund of 2016 to GY; NRF (MSIP; 2015R1A2A2A01003192 and 2015M1A5A1041808) to WS; and an operating grant from the Natural Sciences and Engineering Research Council of Canada to JMA.

Availability of data and materials

The mitochondrial genome datasets in this project has been deposited in the NCBI GenBank database under the accession numbers MG680941, MG680942, MG680943, MG680944, MG680945. The sequence alignments used for phylogenetic analyses have been deposited in the Treebase repository (accession number 22274). Permission to sample from Gomso Bay and Cheongyang pond was obtained from the Korean government. The new strains described in this study are available from the corresponding author or Culture Collections.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Supplementary Materials

Data Availability Statement

The mitochondrial genome datasets in this project has been deposited in the NCBI GenBank database under the accession numbers MG680941, MG680942, MG680943, MG680944, MG680945. The sequence alignments used for phylogenetic analyses have been deposited in the Treebase repository (accession number 22274). Permission to sample from Gomso Bay and Cheongyang pond was obtained from the Korean government. The new strains described in this study are available from the corresponding author or Culture Collections.