Diatom strains and culture conditions

Seven diatom strains from different sources were examined (Table S1). There were no permissions required for those collection sites, and there are no endangered/protected diatoms. All DNA were extracted from cultured materials, several of which are already publicly available. Cerataulina daemon, Roundia cardiophora and Rhizosolenia imbricata were grown in marine f/2 medium [18] in a Percival model I-36LL incubation chamber (Percival, Boone, Iowa, USA) at 21°C; Cyclotella sp. L04_2 and Cyclotella sp. WC03_2 were grown in COMBO medium [19] on a window-lit lab bench; Thalassiosira weissflogii and Chaetoceros simplex were grown in f/2 medium [18] on a window-lit lab bench. The incubator was illuminated with fluorescent lights using a 12:12 hour light:dark photoperiod.

DNA extraction

Diatom cells were pelleted in a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont Company, Newton, CT, USA) for 20 minutes at 7649×g from a culture in the late logarithmic phase of growth. Cells were lysed using a PARR Cell Disruption Bomb (Parr Instrument Company, Moline, IL, USA) filled with nitrogen gas at 1500 psi. Isolation of DNA was performed following Doyle and Doyle [20] with modifications. Cetyl trimethylammonium bromide (CTAB) buffer was augmented with 3% PVP and 3% beta-mercaptoethanol (Sigma, St. Louis MO, USA). Organic phase separation was repeated until the aqueous fraction was clear. DNA pellets were resuspended in ∼200 µL DNase-free water. Following treatment with RNase A (ThermoScientific, Lafayette, CO, USA) samples were again subjected to phase separation with chloroform, and DNA was recovered by ethanol precipitation. Samples were resuspended in DNase-free water, evaluated for concentration by NanoDrop and stored at −20°C.

DNA sequencing and genome assembly

Paired-end (PE) libraries with insert sizes of 400 bp were prepared at the Genome Sequence and Analysis Facility (GSAF) at the University of Texas at Austin. Illumina HiSeq 2000 paired-end platform (Illumina, San Diego, CA, USA) was used to sequence total genomic DNA. The PE Illumina reads were assembled with Velvet v.1.2.08 [21], [22] using multiple k-mers ranging from 71 to 83. Plastid contigs were identified by BLAST analyses of the assembled contigs against published diatom plastid genomes from NCBI. The boundaries between inverted repeats and single copy regions were confirmed bioinformatically or using PCR and Sanger sequencing. The latter two techniques were also utilized to fill gaps in the plastid genome sequences. The PCR primers used for Sanger sequencing were designed by Primer3 [23] in Geneious R6 v.6.1.6 [24] (Table S2).

Genome annotations and analyses

Plastid genomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) [25], followed by manual corrections for start codons using Geneious R6 v.6.1.6. tRNA genes were predicted using DOGMA [25] and tRNAscan-SE 1.21 [26]. Boundaries of rRNA genes, tmRNA ssra gene and signal recognition particle RNA ffs gene were delimited by direct comparison to sequenced diatom orthologues with Geneious R6 v.6.1.6 [24]. Circular plastid genome maps were generated with Organellar GenomeDraw (OGDraw) [27]. Repeated sequences were identified by performing BlastN v.2.2.28+ comparisons of each plastid genome against itself with an e-value cutoff of 1e⁻¹⁰ and at least 90 percent sequence identity. Annotated plastid genomes are available from GenBank using accession numbers KJ958479 – KJ958485. Genome rearrangements were estimated with MAUVE after eliminating one copy of the inverted repeat [28]. Numbers of genome inversions were inferred by GRIMM [29].

Identification of genes transferred to the nucleus and signal peptides

Genes absent from plastid genomes were searched for by BLAST searches in Cyclotella nana nuclear genome against assembled contigs of transcriptome assemblies of T. weissflogii (MMETSP0878) and Rhizosolenia setigera (MMETSP0789) from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) website (http://marinemicroeukaryotes.org/) and nuclear assembly of T. oceanica (http://www.ncbi.nlm.nih.gov/Traces/wgs/?val=AGNL01#contigs) using BLASTN with an e-value cutoff of 1e⁻¹⁰. The previous reported nuclear copy of acp gene in Cyclotella nana (XM_002290970) was used as the query sequence to search for the missing acp genes. SignalP was used to predict signal peptides and cleavage sites [30].

Phylogenetic analysis

Sequences of 20 plastid genes (psaA, psbC, petD, petG, atpA, atpG, rbcL, rbcS, rpoA, rpoB, rps14, rpl33,rnl, rns, ycf89, sufB, sufC, dnaK, dnaB, clpC) from 22 diatom taxa were aligned with MAFFT [31]. This included 15 published diatom plastid genomes and the seven genomes sequenced in this study. All sequences were included, and protein-coding genes were partitioned by gene and codon position. A maximum likelihood tree was constructed with RAxML7.2.8 [32], using the substitution model GTR+G+I and “-f a” option, and 1000 bootstrap replicates were performed to evaluate support for clades.

1. General features of plastid genomes

All seven sequenced plastid genomes mapped as single circles with two IRs dividing the genome into LSC and SSC regions (Figure 1). The genomes are compact and lack introns. The three rRNA subunits (5S, 16S and 23S) are in the IR. Twenty-seven tRNAs together with two other RNAs, transfer-messenger RNA (ssra) and plastid signal recognition particle RNA (ffs), are found in all genomes. Nucleotide composition is highly conserved, with G+C content ranging from 30–32% (Table S3). Four pairs of overlapping genes are present in the seven diatom genomes; sufC-sufB by 1 bp; psbD-psbC by 53 bp; atpD-atpF by 4bp versus 1 bp in Rh. imbricata; and rpl4-rpl23 by 17 bp in the two the Cyclotellas versus 8 bp in the other species (Table S3). The number of protein-coding genes ranges from 122 to 130. All protein-coding genes use the standard plastid-bacterial genetic code except for psbC in Ro. cardiophora, which uses ACG as the start codon instead of ATG. General features of the seven plastid genomes are compared with the two published thalassiosiralean genomes in Table S3.

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Figure 1. Plastid genome maps of seven newly sequenced diatom species.

Species that share the same circular map have the same gene order. Genes on the outside are transcribed clockwise; those on the inside counterclockwise. The ring of bar graphs on the inner circle display GC content in dark grey.

https://doi.org/10.1371/journal.pone.0107854.g001

2. Gene loss

The protein-coding gene complement of the six Thalassiosirales plastid genomes is almost identical with 125 shared genes. A few notable exceptions were found. ycf66 in Ro. cardiophora is a pseudogene as evidenced by several internal stop codons. The acpP1 (acyl carrier protein) gene and the syfB (Phenylalanyl-tRNA synthetase) gene are missing in all Thalassiosirales (Figure 2; Table S4). acpP1 is present in all three sequenced non-Thalassiosirales diatoms; however, syfB is missing only in the more distantly related Rh. imbricata (Figure 2; Table S4). The ycf42 gene is missing in both Ce. daemon and Ch. simplex. The ilvB and ilvH genes, the large and small subunits of acetolactate synthase, are only found in Ce. daemon (Figure 2; Table S4). Several genes are missing from Rh. imbricata, including three photosynthetic genes (psaE, psaI and psaM), the protein translation elongation factor Tu (tufA), syfB and ycf35.

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Figure 2. Phylogeny of Thalassiosirales and other diatom species based on twenty plastid protein-coding genes with gene/intron loss and plastid genome rearrangement events mapped on the branches.

Number of genome inversions within Thalassiosirales were estimated based on Thalassiosirales ancestral genome using GRIMM [29]. Taxa in bold are new genomes sequenced in this study.

https://doi.org/10.1371/journal.pone.0107854.g002

3. Functional gene transfer from plastid to nucleus

One ORF with 83.41% identity to the Cyclotella nana hypothetical plastid targeted acyl carrier protein gene acp3 (XM_002290970) was found in the assembled transcriptome contig (MMETSP0878-20121228|7451_1) of T. weissflogii. The canonical signal peptide cleavage site ASAFVP, same as the signal peptide cleavage site of the acp3 gene in Cyclotella nana, was found and indicated plastid targeting after cleaving between the endoplasmic reticulum (ER) signal peptide and transit peptide (Figure S1). However, SignalP did not indicate the presence of a signal peptide. BLAST analyses of the nuclear acp3 gene of Cyclotella nana against the T. oceanica nuclear genome revealed one ORF with 86.64% identity. The canonical signal peptide cleavage site ASAFAP was found (Figure S1), and SignalP indicated peptide signaling to the ER. Searches for the missing syfB gene using gene sequences from the closely related species Ce. daemon and Ch. simplex against the nuclear genome of Cy. nana and T. oceanica and the transcriptome assembly of T. weissflogii did not identify any matches. Searching the annotated transcriptome data on the MMETSP website of a related species Rhizosolenia setigera Brightwell CCMP 1694 showed several contigs (MMETSP0789-20121207|1125_1, MMETSP0789-20121207|12246-1 etc.) annotated as elongation factor Tu domain or elongation factor Tu binding domain.

4. Genome size and repetitive DNA

The size of the seven sequenced diatom plastid genomes ranges from ∼ 116 kb in Chaetoceros to ∼ 129 kb in Cyclotella (Table S3). Plastid genomes of the Thalassiosirales are larger than the three non-Thalassiosirales species (Ch. simplex, Ce. daemon and Rh. imbricata, Table S3). The sizes of the LSC of the Thalassiosirales are similar to other diatoms sequenced here, however, the sizes of the SSC (24–27 kb) are smaller (27–40 kb) (Figure 3, Table S3). The IRs of Thalassiosirales tend to be larger, ranging from 18 to 23 kb, compared to 7 kb in Ch. simplex and Ce. daemon to 16 kb in Rh. imbricata (Figure 3, Table S3). The plastid genomes are compact with small intergenic spacer regions averaging 87–155 bp (Table S3). BLASTN analysis of each plastid genome against itself revealed only five short tandem repeats in Thalassiosirales with lengths ranging from 79 to 90 bp (Table S5).

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Figure 3. Comparison of inverted repeat boundaries in the seven diatom species newly sequenced for this study plus the two previously sequenced Thalassiosirales.

Tree is that of Figure 2 with previously sequenced outgroup taxa pruned for visual simplicity. The numbers in brown indicate plastid genome size; the numbers in black below each genome fragment indicate the sizes of the LSC, IR and SSC, respectively. Protein coding genes at the IR boundaries are listed in blue. Three red gene blocks are rrn5, rns and rnl, respectively. Names in bold are Thalassiosirales. Underscored names are for taxa newly sequenced for this study.

https://doi.org/10.1371/journal.pone.0107854.g003

The rrnS-trnI-trnA-rnL-rrn5 gene cluster comprises the core of the IR. In Thalassiosirales, genes at the boundaries of IRs and single copy regions are the same, except for T. oceanica, which has an IR expanded through the clpC gene in SSC (Figure 3). The Chaetocerotales (Ch. simplex) and Hemiaulales (Ce. daemon) plastid genomes are smaller than the other diatoms examined. The IR of Ch. simplex is 7403 bp, which is slightly larger than the IR of Ce. daemon at 7004 bp (Figure 3). The IR of Ch. simplex includes one more gene (acpP) than Ce. daemon. The IR of Rhizosoleniales (Rh. imbricata) is larger than Ch. simplex and Ce. daemon.

5. Ancestral plastid genome organization of Thalassiosirales

To reconstruct the ancestral plastid genome organization of Thalassiosirales, shared inversions and ancestral IR/SSC and IR/LSC boundaries were identified. The Mauve alignment identified thirty-two locally collinear blocks (LCBs) shared by the nine diatom plastid genomes examined (Table S6). Gene order within Thalassiosirales is very conserved, except for T. oceanica (Figure 4). Cyclotella nana, T. weissflogii and Ro. cardiophora have identical gene orders. Likewise, Cyclotella sp. L04_2 and Cy. sp. W03_2 have identical gene orders. The gene order of these two groups differs by only a single inversion of five adjacent LCBs (-19)(-15)(-14)(-9)(-10) between rpl 19 and rpl 20 in the LSC region (Table S6; Table S8; Figure 4). The plastid genome of T. oceanica is much more rearranged than other members of Thalassiosirales. GRIMM analysis estimated that ten inversions could explain the different gene orders between Ro. cardiophora and T. oceanica (Figure S2). Based on the most parsimonious reconstruction, the ancestral gene order of Thalassiosirales is the same as that of Ro. cardiophora, T. weissflogii and Cy. nana. The ancestral IR/LSC and IR/LSC boundaries in Thalassiosirales are shared by Ro. cardiophora, T. weissflogii, Cy. nana, Cy. sp. L04_2 and Cy. sp. WC03_2.

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Figure 4. Gene order comparison of the plastid genomes of seven diatoms sequenced for this study plus previously sequenced Thalassiosirales.

Alignments were performed in Geneious R6 [24]with mauveAligner [28]. Taxon names in bold are members of the Thalassiosirales. Names underscored are those sequenced for this study.

https://doi.org/10.1371/journal.pone.0107854.g004

6. Genome rearrangements between Thalassiosirales and the other three diatoms sequenced

Twenty inversions were inferred between the ancestral Thalassiosirales condition and Rh. imbricata (Table S7). Fourteen inversions were inferred between the Thalassiosirales ancestral gene order and Ce. daemon, and seventeen inversions were inferred between the Thalassiosirales ancestral gene order and Ch. simplex (Table S7). Among those inversions two inverted gene blocks, (8) to (-8) and (23) to (-23), are shared by all three non-Thalassiosirales (Figure S3; Table S8). In addition, two inversions, (10)(9) to (-9)(-10) and (30)(31)(32)(27)(26)(25) to (-25)(-26)(-27)(-32)(-31)(-30), are shared by Ce. daemon and Ch. simplex (Figure S3; Table S8). Chaetoceros simplex and Ce. daemon gene orders are more similar to each other than either is to Rh. imbricata (Figure 4, Table S7). The most extensive genome rearrangement occurs between T. oceanica and Rh. imbricata, which differ by twenty-five inversions (Table S7).

Conserved gene content within Thalassiosirales

The plastid genomes of Thalassiosirales have 126–127 protein-coding genes, together with 3 rRNAs and 27 tRNAs (Table S3). Gene content variation is limited in the order with only few notable gene losses/transfers compared to other diatoms (Figure 2). The acpP1 and syfB genes are absent from all Thalassiosirales. It is well known that plastid genes tend to undergo a sequential process of transfer from the plastid to the nucleus [33]. Centralized regulation of plastid metabolism in the nucleus has been suggested as a potential driving force for these transfers [9]. A nuclear encoded plastid targeted acyl carrier protein gene was reported in Cyclotella nana [14] and Synedra acus [13]. Previous research showed that a conserved amino acid motif AXAFXP at the cleavage site of the signal peptide was crucial for plastid targeting [34]. A nuclear encoded, plastid targeted acyl carrier gene was located in the nuclear genomes of T. weissflogii and T. oceanica with a canonical AXAFXP motif (Figure S1). Searching the transcriptome data of Cyclotella meneghiniana from the MMETSP website also revealed an ORF (CAMNT_0012963711) with 84.91% identity with the acp3 gene in Cyclotella nana, and with an ASAFVP signal peptide cleavage motif indicating plastid targeting (data not shown). These results suggest that acpP1 in Thalassiosirales likely represents a functional transfer from the plastid to the nucleus.

Transfer of petF from the plastid to the nucleus is unique to a single species of Thalassiosirales, T. oceanica [12]–[17]. It was suggested that this transfer may have been driven by an adaptation to a low iron environment [9]. To test whether this transfer is environmentally driven or limited to a single species, denser taxon sampling of species throughout the diatom phylogeny in different environments with varying amounts of iron is needed. The sequencing of the plastid genome of Skeletonema, the closest relative of T. oceanica [35], and other diatoms living in the open water with low iron concentration will enhance the understanding of the forces causing the transfer of the petF gene. Another possible gene loss/transfer within Thalassiosirales is ycf66, which is a pseudogene in Ro. cardiophora as suggested by the presence of several internal stop codons. However, more nuclear data are needed to test whether this gene is lost completely or it has been transferred to the nucleus.

Variation of gene content in non-Thalassiosirales species

There are large differences in gene content in non-Thalassiosirales plastid genomes (Figure 2). The large and small subunits of acetolactate synthase, ilvB and ilvH, are reported present in all sequenced red algal plastid genomes [36]. There has been a history of repeated loss of these genes among the 16 diatom genomes [16]. Among the seven new plastid genomes reported here, ilvB and ilvH are absent in all species except Cerataulina daemon. The most parsimonious reconstruction of gene gain/losses suggests that these genes were reacquired independently by this species. More plastid genomes need to be sampled to better understand the loss/gain history of these genes across the diatom tree.

The ycf42 gene is missing from the plastid genomes of both Ce. daemon and Chaetoceros simplex. This gene was reported lost from the plastid genome of Fistulifera sp. JPCC DA0580 [12], Leptocylindrus danicus and Cylindrotheca closterium [16], and has been pseudogenized in the plastid genomes of Asterionellopsis glacialis, Asterionella formosa, Eunotia naegelii and Didymosphenia geminata (Figure 2) [16]. More nuclear genome sequences are needed to determine whether ycf42 has been transferred to the nucleus or has simply been lost.

The ycf35 gene is missing from the Rh. imbricata plastid genome, representing the first case of the loss of this gene from a diatom. The tufA gene, encoding chloroplast protein synthesis elongation factor Tu, is also missing in Rh. imbricata. In the green algal ancestor of land plants, tufA was transferred from the plastid to the nucleus [37]. It is possible that tufA in Rh. imbricata has been functionally transferred to the nucleus but more nuclear data for this species is needed to confirm the transfer.

The most noteworthy gene losses are from the Rh. imbricata plastid genome where the three photosynthetic genes psaE, psaI and psaM are missing. It is well-known that parasitic prokaryotes and eukaryotes have experienced extensive genome size reduction due to loss of genes that are no longer functional [38], [39]. The plastid genome of non-photosynthetic euglenoid flagellate Astasia longa lost all photosynthetic genes from its plastid genome except for rbcL [40]. The non-photosynthetic parasitic flowering plant Epifagus virginiana only contains 42 genes, all genes for photosynthesis and chlororespiration, together with many tRNA and RNA polymerase genes have been lost [41]. But the loss of photosynthetic genes from plastid genomes of non-parasitic plants or algae is rare [42]. There are two possible explanations for the loss of psaE, psaI and psaM from the Rh. imbricata plastid genome. First, these genes may have been functionally transferred to the nucleus. Second, several studies have documented the presence of the endosymbiont, diazotrophic cyanobacterium Richelia intracellularis living within the siliceous frustules of several Rhizosolenia species, including Rh. clevei and Rh. hebetata [43]–[45]. So, it is possible that the missing photosynthetic genes of Rh. imbricata have been horizontally transferred to the endosymbiont, similar to the situation that occurred in the sea slug [46]. However, without nuclear genome/transcriptome data for Rh. imbricata or evidence that a cyanobacterial endosymbiont genome has acquired these genes, it is not possible to determine which of these explanations is more likely.

Genome size

Plastid genome size varies among diatoms, ranging from 116,251 bp in Synedra acus [13] to 165,809 bp in Cylindrotheca closterium [16]. Expansion/contraction/loss of the IR, gene loss and duplication, and reduced size of the introns and intergenic spacer regions are the major factors contributing to variation in genome size [33]. The large genome of Cylindrotheca closterium is mainly due to expanded intergenic spacer regions, which accounts for up to one quarter of the Cylindrotheca plastid genome [16]. It has been previously reported that the larger plastid genome size of T. oceanica compared to the Cyclotella nana is due to the expansion of the inverted repeat [9]. Thalassiosirales have larger plastid genomes than the three sequenced non-Thalassiosirales diatom in this study (Figure 1, Table S3), and most of the diatom species sequenced by Ruck et al. [16]. The low number of repeats and the larger IRs in Thalassiosirales compared other species (Table S3, Figure 3) indicates that their larger genome size is due to expansion of the IR.

Genome rearrangements

Evolutionary events can alter the gene order through inversion, expansion/contraction of the IR, gene duplication/loss, and transposition. Inversions caused by recombination between repeated sequences are considered the major mechanism for gene order changes in plastid genomes [33]. There have been numerous rearrangements among published diatom genomes [16], however, only two species of Thalassiosirales were previously sampled. Completion of plastid genomes of four additional members of the Thalassiosirales and additional diatom species from other lineages shows that gene order within Thalassiosirales is highly conserved with the exception of T. oceanica. The sequenced Thalassiosirales plastid genomes have three different gene order patterns. The first and most common pattern is shared by Ro. cardiophora, T. weissflogii and Cyclotella nana and it represents the ancestral gene order for the order. The second pattern occurs in the two freshwater Cyclotella species, which have one inversion in the LSC region that may be a synapomorphy for this clade (Figure 2, Tables S6–S7). The third pattern is represented by T. oceanica, which is distinct from the rest of the Thalassiosirales. The genome has ten inversions relative to the ancestral genome arrangement for the order (Figure 2, Table S7). The IR boundary of T. oceanica is also distinct from the rest of the Thalassiosirales (Figure 3). IR boundary shifts are a common phenomenon [47] and is likely one of the factors contributing to the extensive rearrangements in T. oceanica. Alverson et al. [35] examined the molecular phylogeny of Thalassiosirales and found that T. weissflogii and Cyclotella species group together, while T. oceanica is more phylogenetically distant from the Thalassiosirales that share similar gene order. To examine whether the gene order change is gradual or punctuated, a wider sampling of plastid genomes across the rest of the Thalassiosirales will be needed to elucidate gene order evolution in this order.