The genome of a nonphotosynthetic diatom provides insights into the metabolic shift to heterotrophy and constraints on the loss of photosynthesis.

Although most of the tens of thousands of diatom species are photoautotrophs, a small number of heterotrophic species no longer photosynthesize. We sequenced the genome of a nonphotosynthetic diatom, Nitzschia Nitz4, to determine how carbon metabolism was altered in the wake of this trophic shift. Nitzschia Nitz4 has retained its plastid and plastid genome, but changes associated with the transition to heterotrophy were cellular-wide and included losses of photosynthesis-related genes from the nuclear and plastid genomes, elimination of isoprenoid biosynthesis in the plastid, and remodeling of mitochondrial glycolysis to maximize adenosine triphosphte (ATP) yield. The genome contains a β-ketoadipate pathway that may allow Nitzschia Nitz4 to metabolize lignin-derived compounds. Diatom plastids lack an oxidative pentose phosphate pathway (oPPP), leaving photosynthesis as the primary source of NADPH to support essential biosynthetic pathways in the plastid and, by extension, limiting available sources of NADPH in nonphotosynthetic plastids. The genome revealed similarities between nonphotosynthetic diatoms and apicomplexan parasites for provisioning NADPH in their plastids and highlighted the ancestral absence of a plastid oPPP as a potentially important constraint on loss of photosynthesis, a hypothesis supported by the higher frequency of transitions to parasitism or heterotrophy in lineages that have a plastid oPPP.

Introduction

Microbial eukaryotes (protists) capture the full breadth of eukaryotic diversity and so exhibit a wide range of trophic strategies that include parasitism, autotrophy, heterotrophy, and mixotrophy, i.e. the ability to acquire carbon and nutrients through both autotrophic and heterotrophic means (Burki et al., 2020). Although the bioenergetics and genes involved in photosynthesis have been well characterized (Falkowski & Raven, 2013), the modes and mechanisms of heterotrophy and mixotrophy vary widely among lineages and, as a result, are more poorly understood (Worden et al., 2015). Many protist lineages (e.g. diatoms and green algae) include a mix of autotrophic, mixotrophic, and/or heterotrophic species (Worden et al., 2015; Bock et al., 2021). Studies focused on evolutionary transitions between nutritional strategies in these groups provide a powerful approach to understanding the ecological drivers, metabolic properties, and genomic underpinnings of different trophic modes in eukaryotes.

Diatoms are photosynthetic algae that account for some 20% of global net primary production (Field et al., 1998). Some diatoms have been shown to supplement their growth through uptake of organic carbon from the environment, making them mixotrophic (Lewin & Lewin, 1967; Hellebust, 1971; Cerón García et al., 2006; Tuchman et al., 2006). Like many plants and algae, some diatoms have lost their ability to photosynthesize (Lewin & Lewin, 1967), but unlike some lineages, such as flowering plants and red algae that have lost photosynthesis many times (Goff et al., 1996; Barkman et al., 2007), loss of photosynthesis has occurred rarely in diatoms, with just two known losses that have spawned only a few dozen species (Frankovich et al., 2018; Onyshchenko et al., 2019). These ‘apochloritic’ diatoms maintain colorless plastids with genomes that are devoid of photosynthesis‐related genes (Kamikawa et al., 2015; Onyshchenko et al., 2019), but their plastids continue to recruit dozens of nuclear‐encoded proteins to service indispensable biosynthetic pathways that remain in the plastid (Kamikawa et al., 2015, 2017; Moog et al., 2020). Retention of the plastid following loss of photosynthesis is not unique to diatoms but is a general pattern in photosynthetic eukaryotes (Hadariová et al., 2018) – even in those rare cases where the plastid genome has been lost (Molina et al., 2014). This highlights the near impossibility of restructuring carbon metabolism in a way that does not include the deeply entrenched cellular compartmentalization that occurred during the establishment of the plastid as an organelle. In addition to understanding why the plastid is retained following loss of photosynthesis, it is important to understand what facilitates or constrains loss of photosynthesis to begin with, and whether such factors vary among the major groups of photosynthetic eukaryotes.

Photosynthesis is central to current models of carbon metabolism in diatoms, where metabolic pathways are highly compartmentalized and where compartmentalization varies among species (Smith et al., 2012). For example, diatom plastids house the Calvin cycle, which is involved in carbon fixation and supplies triose phosphates to anabolic pathways such as biosynthesis of branched‐chain amino acids, aromatic amino acids, and fatty acids (Smith et al., 2012; Kamikawa et al., 2017). The lower payoff phase of glycolysis, which generates pyruvate, adenosine triphosphate (ATP), and NADH, takes place in the mitochondrion (Smith et al., 2012). The centric diatom, Cyclotella nana (formerly Thalassiosira pseudonana; Alverson et al., 2011), has a full glycolytic pathway in the cytosol, but compartmentalization of glycolysis differs in the pennate diatom, Phaeodactylum tricornutum, which lacks a full glycolytic pathway in the cytosol and carries out the lower phase of glycolysis in both the mitochondrion and the plastid (Smith et al., 2012). Compartmentalization helps diatoms avoid futile cycles, which occur when two metabolic pathways work in opposite directions within the same compartment (Smith et al., 2012). All of this requires careful orchestration of protein targeting within the cell, as most carbon metabolism genes are encoded in the nuclear genome (Kroth et al., 2008; Smith et al., 2012). Unlike lineages with primary plastids (Archaeplastida), the secondary plastids of diatoms lack an oxidative pentose phosphate pathway (oPPP) in their plastids (Michels et al., 2005; Gruber et al., 2009), leaving photosystem I (PSI) as the primary source of endogenous NADPH for numerous biosynthetic pathways that take place in diatom plastids.

We sequenced the genome of a nonphotosynthetic diatom in the genus Nitzschia and used the genome to determine how central carbon metabolism was restructured in diatoms that no longer photosynthesize. Based on our findings, we hypothesize that loss of photosynthesis is an easier transition for lineages with a plastid oPPP, and the ancestral presence or absence of a plastid oPPP may play a determinant role in how frequently photosynthesis is lost in different lineages of photosynthetic eukaryotes.

Materials and Methods

Collection and culturing

We collected diatoms from Whiskey Creek, a mangrove‐lined waterway in Dr Von D. Mizell‐Eula Johnson State Park, FL, USA (Onyshchenko et al., 2019). Colorless diatoms were isolated with a Pasteur pipette, and clonal cultures were grown in the dark at 21°C on agar plates made with L1+NPM medium (Guillard, 1960; Guillard & Hargraves, 1993) and 1% Penicillin–Streptomycin–Neomycin solution to retard bacterial growth. We also acquired a photosynthetic Nitzschia (strain CCMP2144) from the National Center for Marine Algae and Microbiota and grew it in L1 medium on a 12 h : 12 h, light : dark cycle.

DNA and RNA sequencing

We sequenced the genome and transcriptome of a single clonal isolate, Nitzschia sp. Nitz4. Cells were removed from agar plates by rinsing with L1 medium, centrifuged, then disrupted with a BeadBeater (BioSpec Products, Bartlesville, OK, USA). Total DNA was extracted with a Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RNA was extracted from Nitzschia Nitz4 and Nitzschia CCMP2144 with a Qiagen RNeasy Kit. Paired‐end, 90 bp reads were sequenced using an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) for 500‐bp DNA and 300‐bp RNA libraries.

Genome and transcriptome assembly

A detailed summary of the genome assembly protocol is available in Supporting Information Methods S1. Briefly, we used Ace (Sheikhizadeh & de Ridder, 2015) to correct sequencing errors, trimmomatic (Bolger et al., 2014) to trim and filter reads, SPAdes (Bankevich et al., 2012) to assemble the genome, and a combination of blobtools (Laetsch & Blaxter, 2017) and the Kmer Analysis Toolkit (Mapleson et al., 2017) to identify and remove assembly contaminants (Methods S1). Contigs were scaffolded using Rascaf (Song et al., 2016) and Sspace (Boetzer et al., 2011), followed by contig extension and gap filling with GapCloser (Luo et al., 2012).

Transcriptome assembly for Nitzschia Nitz4 and Nitzschia CCMP2144 followed Parks et al. (2018). To facilitate comparisons with the published transcriptome of another nonphotosynthetic diatom, Nitzschia NIES‐3581, we downloaded sequence reads from National Center for Biotechnology Information (NCBI) BioProject PRJDB5503 and followed the trimming and assembly methods of Kamikawa et al. (2017).

Genome annotation

A detailed summary of the genome annotation protocol is available in Methods S1. We used Maker (Cantarel et al., 2008) to identify protein‐coding genes in the nuclear genome, using the assembled Nitzschia Nitz4 transcriptome and the proteome of Fragilariopsis cylindrus as evidence to inform Maker gene predictions. We additionally trained and included Augustus (Stanke et al., 2008) and Snap (Korf, 2004) models for ab initio gene prediction. In our search for genes related to carbon metabolism, we manually annotated several genes that were not in the final set of Maker predictions. Functional information was added to the predicted gene set by searching the SwissProt and UniProt Reference Proteomes databases with Ncbi‐blastp (Camacho et al., 2009). Protein domains were identified through InterProScan searches (Jones et al., 2014) against the Pfam (El‐Gebali et al., 2019) and PANTHER (Thomas et al., 2003) databases. We used Tmhmm (Krogh et al., 2001) to identify transmembrane helices and characterized coding and noncoding RNAs using a combination of tRNAscan‐SE (Chan & Lowe, 2019), RNAmmer (Lagesen et al., 2007), and Infernal (Nawrocki & Eddy, 2013) against the Rfam database (Kalvari et al., 2021).

Estimation of heterozygosity

Genome‐wide heterozygosity was estimated by mapping trimmed reads to the genome with Bwa‐mem (Li & Durbin, 2009) and calling variants with the Genome Analysis Toolkit (Gatk) HaplotypeCaller tool (DePristo et al., 2011; Poplin et al., 2018). We filtered variants according to Gatk’s Best Practices and by approximate read depth. Filtered variants were then evaluated using Gatk’s VariantEval tool and annotated with SnpEff (Cingolani et al., 2012). Additional details are provided in Methods S1.

Gene family evolution

We used orthofinder (Emms & Kelly, 2019) to construct clusters of orthologous protein‐coding genes and infer the species phylogeny, using Mafft (Katoh & Standley, 2013) for multiple sequence alignment and fasttree (Price et al., 2010) for gene tree reconstruction. Sequencing errors, allelic variants, and splice isoforms can result in transcriptome assemblies that contain many more transcripts than genes in the genome (Ono et al., 2015). The large number of redundant transcripts in the transcriptomes of Nitzschia CCMP2144 and Nitzschia NIES‐3581 led to artifactual patterns of gene family expansion and contraction (Supporting Information Fig. S1), so our analyses of gene family evolution only included species with sequenced genomes. To more precisely characterize gene presence/absence in Nitzschia Nitz4, we ran a separate OrthoFinder analysis that included transcriptomes from Nitzschia CCMP2144, Nitzschia NIES‐3581, and an additional outgroup, Bolidomonas pacifica (Kessenich et al., 2014).

We used Cafe (Han et al., 2013) to characterize patterns of gene family evolution across 6092 gene families identified by OrthoFinder, excluding gene families where copy numbers varied by > 100 between species or that were not present in the last common ancestor of diatoms. Cafe uses a phylogenetic tree, gene family sizes, and a birth–death model to identify gene families and branches on the species tree that have experienced statistically significant, rather than stochastic, changes in gene family size (Hahn et al., 2005). We included error models that accommodated errors in genome assembly and annotation. The birth–death rate (λ) was estimated both in a global model with a single parameter as well as a three‐rate model with separate parameters for the outgroups, Nitzschia Nitz4, and other diatoms. The three‐rate λ model provided the best fit to the data. Rapidly expanding or contracting gene families were considered significant at the default P < 0.01.

Characterization of carbon metabolism genes

We manually annotated and characterized genes with known roles in carbon metabolism. To do so, we began with a set of core carbon metabolic genes from diatoms (Smith et al., 2012; Kamikawa et al., 2017) as search terms to download related genes from NCBI’s nr database (release 223.0 or 224.0), filtered to include RefSeq accessions only. We expanded our searches as necessary to characterize each gene in major carbon metabolic pathways. The Transporter Classification Database (TCDB; Saier et al., 2014) was used to identify carbon transporters, and the Carbohydrate‐Active Enzymes (CAZy) database (Lombard et al., 2014) was used to identify genes related to saccharide degradation.

Prediction of protein localization

We used SignalP (Petersen et al., 2011), ASAFind (Gruber et al., 2015), ChloroP (Emanuelsson et al., 1999), TargetP (Emanuelsson et al., 2007), MitoProt (Claros, 1995), and Hectar (Gschloessl et al., 2008) to predict whether proteins were targeted to the plastid, mitochondrion, cytoplasm, or endoplasmic reticulum (ER) for the secretory pathway. Following Traller et al. (2016), proteins that SignalP, ASAFind, and Hectar predicted to contain a plastid signal peptide were classified as plastid‐targeted. Proteins predicted as mitochondrial‐targeted by any two of TargetP, MitoProt, and Hectar were classified as localized to the mitochondrion. Proteins in the remaining set were classified as ER‐targeted if they had ER signal peptides predicted by SignalP and Hectar.

Results and Discussion

Nitzschia sp. strain Nitz4

We collected and isolated Nitzschia Nitz4 (see micrographs in Supporting Information Fig. S2) from a shallow, warm, mangrove‐lined coastal waterway in the south‐eastern United States. Our sample included near‐surface plankton, submerged (< 1 m) sand, and nearshore unsubmerged sand. Based on our collection, previous reports of these species in near‐surface plankton (Blackburn et al., 2009), and the high degree of active motility we observed in our culture, it is likely that Nitzschia Nitz4 is tychoplanktonic, i.e. living principally in the benthos but circulating in the shallow plankton. Previous phylogenetic analyses placed Nitzschia Nitz4 within a larger clade of nonphotosynthetic Nitzschia (Onyshchenko et al., 2019). Nitzschia Nitz4 grew in the presence of antibiotics for approximately 1 yr, but the strain experienced gradual size diminution, which is characteristic of diatoms (Edlund & Stoermer, 1997), and ultimately died after failing to reproduce sexually and reconstitute its maximal size. This suggests that, like other members of the Bacillariales (Levialdi Ghiron et al., 2008), Nitzschia Nitz4 was dioecious.

Plastid genome reduction

As described previously (Onyshchenko et al., 2019), the plastid genome of Nitzschia Nitz4 experienced near‐complete loss of intergenic DNA and wholesale loss of genes encoding the photosynthetic apparatus (Supporting Information Table S1). The plastid genome of Nitzschia Nitz4 contains just 90 of the 122 core genes present in photosynthetic diatoms (Ruck et al., 2014). The plastid genome also experienced a substantial downward shift in GC content, from > 30% in photosynthetic species to 22.4% in Nitzschia Nitz4 (Table S1), a common pattern in streamlined bacterial genomes (Giovannoni et al., 2014) that may reflect mutational bias (Hershberg & Petrov, 2010) or nitrogen frugality in response to its limitation (GC base pairs contain more nitrogen than AT base pairs; Dufresne et al., 2005).

Nuclear genome characteristics

The genome was assembled into 1015 scaffolds totaling 27.6 Mb in length and an average coverage depth of 221 reads. The assembly was highly contiguous: the largest scaffold was 479 884 bp in length, and half the genome was contained in 88 scaffolds, each one longer than 91 206 bp (assembly N50; Table S2). These assembly statistics compare favorably to other diatom genomes, including ones sequenced with long‐read technologies (Table S2). The Nitzschia genome is similar in GC content (48%) to other diatom genomes, indicating that the decrease in GC content experienced by the plastid genome (Table S1) did not extend to its nuclear and mitochondrial (Guillory et al., 2018) genomes.

We identified 9464 gene models and 14 alternative isoforms, for a total of 9478 predicted protein genes in Nitzschia Nitz4 (Table S2). Most gene models (9274) were supported by transcriptome or protein similarity evidence. To estimate genome completeness, we used Busco to count the number of conserved protein‐coding genes in the genome (Simão et al., 2015). Although Nitzschia Nitz4 has the fewest genes of all diatom genomes sequenced to date, its Busco count matches or exceeds other diatom genomes (Table S2). Most of the predicted proteins (86%) had significant Blast hits (e‐value < 1e‐6) to SwissProt or UniProt, and 66% contained significant InterProScan hits (e‐value < 1e‐5) to protein domains in the Pfam or PANTHER databases.

We characterized single nucleotide polymorphisms (SNPs) and small structural variants in the genome of Nitzschia Nitz4, full details of which are provided in Table S3. The estimated genome‐wide heterozygosity was 0.0028 and included 13 410 synonymous and 9958 nonsynonymous SNPs in protein‐coding genes (Table S3). Nitzschia Nitz4 had a higher proportion of nonsynonymous to synonymous substitutions (0.74) than was reported for the model diatom, Phaeodactylum tricornutum (0.28–0.43) (Krasovec et al., 2019; Rastogi et al., 2020), and the green alga, Ostreococcus tauri (0.2) (Blanc‐Mathieu et al., 2017).

Gene family evolution and signatures of heterotrophy

The complete genome sequence of Nitzschia Nitz4 allowed us to characterize gene family expansions, contractions, and losses related to the transition to heterotrophy (Fig. 1). Ortholog clustering showed that most genes in Nitzschia Nitz4 were shared with other diatoms, with 8285 (87.5%) of the Nitzschia Nitz4 genes assigned to orthogroups that included other diatoms. The remaining 1179 genes were unique to Nitzschia Nitz4, and most of these (860) were singletons. The number of Nitz4‐specific genes decreased to 374 when the transcriptomes of Nitzschia NIES‐3581 and Nitzschia CCMP2144 were considered. This number (374) is still probably an overestimate of the unique gene content given the greater recovery of genes in the Nitzschia Nitz4 genome (99% stramenopile Buscos) compared to the Nitzschia NIES‐3581 transcriptome (89% stramenopile Buscos) (Table S2).

Fig. 1

Rapidly evolving gene families in Nitzschia Nitz4. (a) The histogram shows the total count of gene families for each combination of species; only those combinations/intersections containing ≥ 2 gene families are shown. Each row in the upset plot (bottom) represents a diatom or outgroup species, and the total number of intersecting gene families follows the species name. Vertical black lines connecting rows show which species contain the rapidly evolving gene families in a given intersection (i.e. category in the histogram). As an example, the far‐left column shows 46 rapidly evolving gene families, and the upset plot below shows that those 46 families are shared by all 11 species. Intersections containing gene families that have contracted or expanded significantly in Nitzschia Nitz4 are colored orange or blue, respectively. (b) Each column represents a diatom or outgroup species, and each row a rapidly evolving gene family in Nitzschia Nitz4, identified by its orthogroup identifier and annotation. Circle size is proportional to gene family size, whereas the color of the circle shows whether the gene family was significantly expanded (blue) or contracted (orange) in Nitzschia Nitz4.

Although the plastid genome experienced the greatest reduction in gene content, the nuclear genome also experienced losses of genes that are otherwise highly conserved across diatoms, based on their shared presence across most or all of the other taxa in our analysis (Fig. 1). Among the 20 rapidly evolving gene families in Nitzschia Nitz4 identified by Cafe, most of these (15/20) were contractions, and 10 of the contractions involved gene families that were fully and uniquely lost in Nitzschia Nitz4 (Fig. 1b). Many of the gene families lost in Nitzschia Nitz4 are related directly or indirectly to photosynthesis. For example, Nitzschia Nitz4 is devoid of gene families associated with pigmentation and light harvesting for photosynthesis (Fig. 1b). Calcium is an important regulator of photosynthesis in plants and algae (Hochmal et al., 2015), and the upregulation of plastid‐localized calmodulin‐dependent protein kinases at low CO2 in C. nana suggested a role for calcium‐dependent signaling in the regulation of photosynthesis in diatoms as well (Clement et al., 2017). Loss of the calmodulin‐lysine N‐methyltransferase family of proteins in Nitzschia Nitz4 (Fig. 1b) suggests that its function is photosynthesis‐related in diatoms and, consequently, no longer necessary in the absence of photosynthesis. Nitzschia Nitz4 also lost a family of protein translocases that import nuclear‐encoded proteins into the plastid (Fig. 1b), as well as serine proteinase (Fig. 1b), a protein that facilitates turnover of photodamaged photosystem II (PSII) subunits in the thylakoid membrane (Nagao et al., 2012). Finally, although not highlighted by the Cafe analysis because it involved loss of a single‐copy gene, Nitzschia Nitz4 no longer has a 2‐keto‐3‐deoxyphosphogluconate aldolase (EDA) gene, which altered mitochondrial glycolysis. Possible causes and consequences of this loss are discussed in the section entitled, ‘Loss of the mitochondrial Entner–Doudoroff glycolytic pathway’.

Central carbon metabolism

Carbon metabolism in heterotrophs

Classically, the major pathways for carbon metabolism in heterotrophic organisms include glycolysis, the oPPP, and the tricarboxylic acid (TCA, or Krebs) cycle. Glycolysis uses glucose (or other sugars that can be converted to glycolytic intermediates) to generate pyruvate and ATP. Pyruvate serves as a key ‘hub’ metabolite that can be used for amino acid biosynthesis, anaerobically fermented to lactate or ethanol, or converted into another hub metabolite, acetyl‐ coenzyme A (acetyl‐CoA). Acetyl‐CoA can then be used for fatty acid biosynthesis or can be further oxidized via the Krebs cycle to generate ATP, reducing equivalents (FADH2 and NADH), and certain amino acid precursors. The oPPP runs parallel to glycolysis, but unlike glycolysis, is largely anabolic, generating ribose‐5‐phosphate (a nucleotide precursor) and the key reducing agent, NADPH. When the carbon and energy demands of a cell have been met, gluconeogenesis converts pyruvate back into glucose, which can be converted into storage carbohydrates (e.g. chrysolaminarin and trehalose; Beattie et al., 1961; Villanova et al., 2017) as well as glycolytic intermediates for anabolism (Smith et al., 2012; Tymoczko et al., 2013). Because gluconeogenesis is essentially the reverse of glycolysis and shares many of the same enzymes, flux through each pathway is regulated through the control of specific enzymes driving irreversible reactions (GK, PFK, PK, and FBP).

Oxidative pentose phosphate pathway

In diatoms, flux through central metabolism is also controlled through compartmentalization of different portions of key metabolic pathways. For example, in diatoms the oxidative branch of the PPP, which reduces NADP+ to NADPH, is only present in the cytoplasm, whereas an incomplete nonoxidative PPP is present in the plastid, thereby limiting the available sources of NADPH in the plastid to PSI, sugar phosphates imported from the cytosol (Michels et al., 2005; Moog et al., 2020), and as detailed later, possibly a plastid NAD(P) transhydrogenase (NTH) that interconverts NADH to NADPH. As a result, nonphotosynthetic diatoms lack two principal sources of plastid NADPH – PSI and the oPPP – and therefore require alternative ways to reduce NADP+ in their plastids, which continue to house essential NADPH‐consuming biosynthetic pathways (e.g. fatty acid and amino acid biosynthesis; Fig. 2). Moreover, diatom plastids export reducing equivalents to the mitochondria in exchange for ATP (Bailleul et al., 2015), potentially further depleting NADPH levels in the plastid. Photosynthetic diatoms exposed to low or no light likely face a similar NADPH deficit (Grouneva et al., 2009; Gruber et al., 2009; Mekhalfi et al., 2014; Kim et al., 2016; Gruber & Kroth, 2017), which suggests that the ability to continue reducing NADP+ in their plastids in the absence of photosynthesis may be a general challenge for diatoms.

Fig. 2

Genome‐based model of carbon metabolism in the plastid (left) and cytosol (right) of Nitzschia Nitz4. Chemical substrates and products are shown in blue, enzymes encoded by genes present in Nitzschia Nitz4 are shown in dark yellow, and cofactors are shown in green. Black and yellow arrows show predicted reactions, pathways, or direction of transport of chemical substrates across membranes. Polygons show presence (pink) or absence (white) of complete pathways. Missing genes and pathways are shown in white.

Glycolysis in Nitzschia Nitz4

We developed a model of central carbon metabolism to determine whether the switch to heterotrophy in Nitzschia Nitz4 included changes beyond the loss of photosynthesis‐related genes. Cytosolic glycolysis in Nitzschia Nitz4 is similar to photosynthetic diatoms (Table S4). Like other raphid pennate diatoms, Nitzschia Nitz4 is missing cytosolic enolase, which catalyzes the penultimate step of glycolysis, suggesting that Nitzschia Nitz4 lacks a full cytosolic glycolysis pathway (Fig. 2; Table S4). Nitzschia Nitz4 does have plastid‐ and mitochondrial‐targeted enolase genes, indicating that later stages of glycolysis occur in these compartments (Figs 2, 3; Table S4). Nitzschia Nitz4 has two copies of the gene encoding phosphoglycerate kinase (PGK), a key enzyme that catalyzes a reversible reaction used in both glycolysis and gluconeogenesis. Enzymes involved in the latter half of glycolysis are localized to the mitochondrion in most diatoms (Kroth et al., 2008; Smith et al., 2012), but neither PGK gene in Nitzschia Nitz4 contains a mitochondrial targeting signal – instead, one copy is predicted to be plastid‐targeted and the other cytosolic (Fig. 2; Table S4). As discussed later, while plastid targeting of PGK may be important for reducing NADP+, it is likely that one or both of the plastid and cytosolic PGK genes are dual‐targeted to the mitochondrion to complete the latter half of the pathway. Dual‐targeting of proteins to the plastid and mitochondrion has been demonstrated in other diatoms (Gile et al., 2015).

Fig. 3

Genome‐based model of carbon metabolism in the mitochondria of Nitzschia Nitz4. Membrane proteins are shown in light yellow, chemical substrates and products are shown in blue, enzymes encoded by genes present in Nitzschia Nitz4 are shown in dark yellow, cofactors are shown in green, and protons in orange. Arrows show predicted reactions, pathways, or direction of transport of chemical substrates across membranes. The missing Entner–Doudoroff glycolytic pathway is shown in white.

Pyruvate hub

Because pyruvate is a key central metabolite that can be used for both catabolism and anabolism, we characterized the presence and localization of ‘pyruvate hub enzymes’ (Smith et al., 2012) in Nitzschia Nitz4 (Table S4). Nitzschia Nitz4 has lost several pyruvate conversion genes, most dramatically in the plastid. For example, the pennate diatom Phaeodactylum tricornutum uses pyruvate phosphate dikinase (PPDK) to perform the first step of gluconeogenesis in the plastid, whereas the multipolar centric diatom C. nana uses phosphoenolpyruvate synthase (PEPS) (Smith et al., 2012). Nitzschia Nitz4 lacks plastid‐localized copies of both enzymes (Table S4). Additionally, and in contrast to photosynthetic diatoms, Nitzschia Nitz4 lacks a plastid‐localized pyruvate carboxylase. Together, these results suggest that Nitzschia Nitz4 has lost the ability to initiate gluconeogenesis in the plastid and instead relies on mitochondrial‐localized enzymes to determine the fate of pyruvate (Figs 2, 3; Table S4). The mitochondrial‐localized pyruvate hub enzymes of photosynthetic diatoms are mostly present in Nitzschia Nitz4 (Fig. 3), with the sole exception being malate decarboxylase (or malic enzyme (ME)), which converts malate to pyruvate and CO2 (Kroth et al., 2008; Smith et al., 2012). In C4 plants, ME is used to increase carbon fixation at low CO2 concentrations (Sage, 2004). If ME performs a similar function in diatoms, then it may be dispensable in nonphotosynthetic species.

Loss of isoprenoid biosynthesis in the plastid

Many photosynthesis‐related molecules such as chlorophylls, carotenoids, and plastoquinones are derivatives of isoprenoids that are produced by the mevalonate (MVA) pathway in the cytosol or, in plastids, by the methylerythritol 4‐phosphate (MEP) pathway, also known as the nonmevalonate pathway. The absence of an MVA pathway in apicomplexans, chlorophytes, and red algae suggests that it is not necessary to maintain both the MVA and MEP pathways for isoprenoid biosynthesis (Rohmer, 1999). The genome of Nitzschia Nitz4 has lost its plastid MEP pathway (Fig. 2), indicating an inability to synthesize carbon‐ and NADPH‐consuming isoprenoids, the precursors of photosynthetic pigments, in the plastid. The transcriptome of Nitzschia NIES‐3581 is missing a plastid MEP pathway as well (Kamikawa et al., 2017).

Loss of the mitochondrial Entner–Doudoroff glycolytic pathway

The loss of photosynthesis in Nitzschia Nitz4 underscored the energetic link between the plastids and mitochondria of diatoms (Bailleul et al., 2015). Diatoms use a prokaryotic‐like Entner–Doudoroff (ED) pathway for mitochondrial glycolysis (Fabris et al., 2012). The ED glycolytic pathway is characterized by two key reactions, the first of which is catalyzed by 6‐phosphogluconolactone dehydratase (EDD). Although Nitzschia Nitz4 lacks a mitochondrial‐targeted gene for EDD (Fig. 3), this was not different from other diatoms, most of which have an EDD with a plastid, not mitochondrial, targeting prediction. Although the first reaction in the ED pathway was previously predicted to occur in the mitochondrion (Fabris et al., 2012), our findings suggest that in diatoms EDD is either dual‐targeted to the plastid and mitochondrion, or that the first reaction in the ED pathway occurs in the cytosol.

The EDA enzyme catalyzes the second reaction in the ED pathway and was present in all of the photosynthetic diatoms in our analysis, including a photosynthetic species from the genus Nitzschia (CCMP2144). The EDA gene was missing, however, from both nonphotosynthetic diatoms – Nitzschia Nitz4 (Fig. 3) and Nitzschia NIES‐3581 – indicating that its loss was recent and likely related to the loss of photosynthesis. Loss of EDA remodeled mitochondrial glycolysis in these species, transitioning them from the ancestral and highly conserved ED pathway to the more classic Embden–Meyerhof–Parnas (EMP) glycolytic pathway (Fig. 3).

The ED glycolytic pathway generates half the ATP as the EMP pathway but requires production of much less protein to achieve the same flux (Flamholz et al., 2013). As a result, these two strategies reflect a trade‐off between energy yield and production costs of glycolytic enzymes (Flamholz et al., 2013). Organisms such as phototrophs that have abundant nonglycolytic sources of ATP but face limiting nitrogen commonly possess the ED pathway (Flamholz et al., 2013). We hypothesize that the loss of photosynthesis exerts strong selection to shift from mitochondrial ED to EMP glycolysis. The presence of an ED pathway in many autotrophic, but not heterotrophic, stramenopiles (Río Bártulos et al., 2018) supports this hypothesis, and suggests that the transition from an ED to EMP pathway in secondary heterotrophs may be a general phenomenon.

Diatoms plastids likely export and shuttle reducing equivalents to the mitochondria in exchange for ATP (Bailleul et al., 2015), so the switch to a mitochondrial EMP pathway would not only increase ATP yield in the mitochondrion, but it might also increase ATP flux into plastids where ATP is probably in short supply in nonphotosynthetic diatoms. NADPH might also be scarce in the plastids of nonphotosynthetic diatoms, which need to preserve an optimal ATP : NADPH ratio through decreases in demand (e.g. by loss of the MEP) and modulation of ATP production in the mitochondrion. In addition to generating half the ATP, the ED and EMP pathways also differ in their redox cofactors. In diatoms, the ED pathway yields one NADPH in the cytosol and one NADH in the mitochondrion (Fabris et al., 2012), whereas the EMP pathway yields two NADH in the mitochondrion. As a result, the transition from an ED to EMP pathway eliminated a source of cytosolic NADPH, which is noteworthy given the potential importance of NADPH limitation in the plastid (see section entitled ‘NADPH limitation and constraints on the loss of photosynthesis’).

Carbon active enzymes

The transition to heterotrophy in Nitzschia Nitz4 did not appear to be facilitated by acquisitions of novel carbon transporters or carbohydrate‐cleaving enzymes, or by significant expansions of these gene families (Fig. 1; Tables S5, S6).

A β‐ketoadipate pathway in diatoms

One of our primary goals was to develop a comprehensive model of central carbon metabolism for nonphotosynthetic diatoms. A systematic search for carbon‐active enzymes revealed a β‐ketoadipate pathway, whose primary function is degradation of plant‐derived aromatic substrates (Harwood & Parales, 1996). The β‐ketoadipate pathway is typically associated with soil‐dwelling fungi and eubacteria and consists of two main branches, one of which converts protocatechuic acid (protocatechuate, or PCA) and the other catechol, into β‐ketoadipate (Harwood & Parales, 1996). Diatoms possess the PCA branch of the pathway (Fig. 3; Table S7). Intermediate steps of the PCA branch differ between fungi and bacteria (Harwood & Parales, 1996), and diatoms possess the bacterial‐like PCA branch. In the final steps of the pathway, β‐ketoadipate is converted to the TCA intermediates succinyl‐CoA and acetyl‐CoA (Fig. 3). The β‐ketoadipate pathway is not unique to Nitzschia Nitz4 as most or all of the requisite genes are present in other sequenced diatom genomes and transcriptomes, which suggests that the pathway is an ancestral feature of diatom genomes (Table S8).

We initially discovered eight genes in Nitzschia Nitz4 with matches to bacterial‐like proteins annotated as intradiol ring‐cleavage dioxygenase or protocatechuate 3,4‐dioxygenase (P3,4O). This is the first enzyme in the protocatechuate branch of the β‐ketoadipate pathway and is involved in the degradation of aromatic compounds through intradiol ring cleavage of PCA (Harwood & Parales, 1996). Fission of the highly stable aromatic ring structure of PCA is the most difficult step of the pathway (Fuchs et al., 2011), so it is noteworthy that both nonophotosynthetic Nitzschia species are enriched for this gene – each possessing at least eight copies – whereas other diatoms have four or fewer copies (Table S8). Expansion of the P3,4O gene family in Nitzschia Nitz4 and NIES‐3581 suggests that nonphotosynthetic diatoms might be more efficient at carrying out the putative bottleneck reaction of the β‐ketoadipate pathway.

Targeted searches of the annotated genes and scaffolds revealed genes for subsequent steps of the pathway, including 3‐carboxymuconate lactonizing enzyme (CMLE), 4‐carboxymuconolactone decarboxylase (CMD), and β‐ketoadipate enol‐lactone hydrolase (ELH) (Fig. 3; Table S7). Enzymes encoding the final two steps, 3‐ketoadipate:succinyl‐CoA transferase (TR) and 3‐ketoadipyl‐CoA thiolase (TH), are missing from Nitzschia Nitz4. However, these enzymes are similar in both sequence and predicted function – attachment and removal of CoA – to two mitochondrial‐targeted genes that are present in Nitzschia Nitz4, succinyl‐CoA:3‐ketoacid CoA transferase (SCOT) and 3‐ketoacyl‐CoA thiolase (3‐KAT), respectively (Parales & Harwood, 1992; Kaschabek et al., 2002). Finally, all sequenced diatom genomes, including Nitzschia Nitz4, have a putative 4‐hydroxybenzoate transporter (PcaK; Table S8) that reportedly can import PCA into the cell (Nichols & Harwood, 1997).

Nonphotosynthetic Nitzschia are strictly marine and found on coastlines, and they are especially common in mangrove habitats (Ishii & Kamikawa, 2017; Onyshchenko et al., 2019). Vascular plants, including mangroves, are a major source of dissolved organic carbon (DOC) in coastal ocean ecosystems (Moran et al., 1991b; Moran & Hodson, 1994b; Hedges et al., 1997). Plant‐derived DOC includes lignin and a suite of lignin‐derived phenols that support secondary production of diverse communities of marine bacteria (Moran & Hodson, 1994a; Mou et al., 2008). Vanillin is one of the principal phenolic subunits of lignin and comprises a large fraction of the total lignin phenols in some systems (Kattner et al., 1999; Cotrim da Cunha et al., 2001). One of the products of vanillin degradation, PCA, is the predicted substrate for the β‐ketoadipate pathway (Fig. 3), which serves as a ‘funnel’ for numerous lignin‐derived PCA or catechol precursors (Harwood & Parales, 1996; Fuchs et al., 2011). Although bacteria drive biodegradation of lignin phenols in the ocean, the presence of a β‐ketoadipate pathway in diatoms suggests they may play a role in this part of the ocean carbon cycle as well.

Although Nitzschia Nitz4 and other diatoms possess a full β‐ketoadipate pathway, the genomic prediction – that diatoms with this pathway metabolize naturally occurring aromatic compounds into products (succinyl‐ and acetyl‐CoA) that feed into the TCA cycle – requires experimental corroboration. It will be important to show whether diatoms import PCA, whether it augments growth, and whether the terminal products are converted into cellular biomass or, alternatively, whether the pathway is for catabolism of PCA or a related compound. The piecemeal β‐ketoadipate pathway in some diatoms raises questions about the functions of these genes and whether the complete pathway is restricted to diatoms in coastal areas where lignin‐derived phenols are most abundant (Moran et al., 1991a; Opsahl & Benner, 1997).

NADPH limitation and constraints on the loss of photosynthesis

Plastids are the site of essential biosynthetic pathways and, consequently, remain biochemically active following loss of their primary function, photosynthesis (Neuhaus & Emes, 2000). Most biosynthetic pathways in the plastid require the reducing cofactor, NADPH, which is generated principally through the light‐dependent reactions of photosynthesis and the oPPP. The primary‐plastid‐containing Archaeplastida – including glaucophytes (Fester & Schnenk, 1997), green algae (Weber & Linka, 2011), land plants (Schnarrenberger et al., 1995), and red algae (Moriyama et al., 2014) – have both cytosolic‐ and plastid‐localized oPPPs. In plants, the plastid oPPP generates NADPH reductants in nonphotosynthetic embryos (Andriotis & Smith, 2019) and root tissues (Bowsher et al., 1992), and in Archaeplastida, the plastid oPPP continues to reduce NADP+ when photosynthesis is lost – something that has occurred at least five times in green algae (Rumpf et al., 1996; Tartar & Boucias, 2004), nearly a dozen times in angiosperms (Barkman et al., 2007), and some 100 times in red algae (Blouin & Lane, 2012). Chrysophyte algae, which also have a plastid oPPP, have lost photosynthesis at least seven times (Dorrell et al., 2019). Despite their age (200 Myr) and estimates of species richness on par with angiosperms and greatly exceeding green and red algae, diatoms are known to have lost photosynthesis only twice (Frankovich et al., 2018; Onyshchenko et al., 2019).

Unlike Archaeplastida, diatoms lack a plastid oPPP and therefore rely primarily on photosynthesis for the reduction of NADP+ in their plastids (Michels et al., 2005; Gruber et al., 2009). Nevertheless, the plastids of nonphotosynthetic diatoms continue to be the site of numerous NADPH‐consuming reactions (Fig. 2; Kamikawa et al., 2017). Without an oPPP, it is unclear how diatom plastids meet their NADPH demands during periods of prolonged darkness or, by extension, in species that have lost photosynthesis. Possible enzymatic sources include an NADP‐dependent isoform of the glycolytic enzyme GAPDH or conversion of malate to pyruvate by ME (Smith et al., 2012). In Nitzschia Nitz4, NADP‐GAPDH is predicted to be targeted to the periplastid compartment, but ME appears to have been lost.

In addition to enzymatic production, plastids might acquire NADPH via transport from the cytosol, but transport of large polar molecules like NADPH through the inner plastid membrane might be prohibitive (Taniguchi & Miyake, 2012). Arabidopsis has a plastid NAD(H) transporter (Palmieri et al., 2009), but to our knowledge no NADP(H) transporters have been identified in diatoms. Cells commonly use ‘shuttle’ systems to exchange redox equivalents across intracellular membranes (Noctor et al., 2011). Although these systems generally shuttle excess reductants from the plastid to the cytosol (Taniguchi & Miyake, 2012), the triose phosphate/3‐phosphoglycerate shuttle is hypothesized to run in reverse to generate NADPH in the plastids of nonphotosynthetic diatoms (Moog et al., 2020) as well as the apicoplast of the malaria parasite, Plasmodium falciparum (Lim & McFadden, 2010). In this model, triose phosphates are transferred from the cytosol to the plastid in exchange for 3‐phosphoglyceric acid (PGA), resulting in the generation of ATP and NADPH in the plastid by PGK and NADP‐GAPDH (Fig. 4; Moog et al., 2020). Nitzschia Nitz4 contains a plastid‐targeted NADP‐GAPDH and two copies of PGK (one cytosolic and one plastid‐localized). This differs from photosynthetic diatoms, where at least one PGK is localized to the mitochondrion (Smith et al., 2012).

Fig. 4

Possible sources of NADPH in the plastid of Nitzschia Nitz4. The ‘reverse’ triose phosphate/3‐phosphoglycerate shuttle would exchange cytoplasmic NADH for NADPH (Moog et al., 2020), and NAD(P) transhydrogenase (NTH) would use the proton gradient generated by adenosine triphosphate (ATP) hydrolysis, via ATP synthase (Kamikawa et al., 2015), to catalyze interconversion of NADH to NADPH. Chemical substrates and products are shown in blue, protons in orange, and cofactors are shown in green. Arrows show predicted reactions or direction of transport of protons or chemical substrates across membranes. In addition to ATP synthase and NTH, other enzymes encoded by genes present in Nitzschia Nitz4 are shown in dark yellow.

An apicoplast membrane‐targeted NTH was recently discovered in Plasmodium (Saeed et al., 2020). Normally localized to the mitochondrial membrane in eukaryotes, NTH catalyzes, via hydride transfer, the interconversion of NADH and NADPH (Fig. 4; Jackson et al., 2015). The apicoplast‐targeted NTH might provide a novel and necessary source of NADPH to that organelle (Lim & McFadden, 2010). Nitzschia Nitz4 has two NTH genes, one targeted to the mitochondrion (NITZ4_001957) and a second (NITZ4_000738) to the plastid. Although optimized for Viridiplantae, SChloro (Savojardo et al., 2017) predicted that NITZ4_000738 is localized to the thylakoid membrane (Fig. 4). In addition to facilitating translocation of proteins into the thylakoids (Kamikawa et al., 2015), the proton gradient created through ATP hydrolysis might also reduce NADP+ in the plastid. If NTH is indeed involved in the reduction of NADP+, this highlights another way in which nonphotosynthetic diatoms and apicomplexans converged upon similar solutions to NADPH limitation in their plastids.

Indispensable pathways in diatom plastids include fatty acid biosynthesis, branched chain and aromatic amino acid biosynthesis, and Asp‐Lys conversion – all of which consume carbon and NADPH (Fig. 2). In the absence of photosynthesis for carbon fixation and reduction of NADP+, and without an oPPP to reduce NADP+ in the plastid, essential building blocks and reducing cofactors for biosynthetic pathways in the plastid are probably in short supply in nonphotosynthetic diatoms. The need to conserve carbon and NADPH likely drove loss of the isoprenoid‐producing MEP from the plastid in nonphotosynthetic diatoms (Kamikawa et al., 2017), chrysophytes (Dorrell et al., 2019), and euglenophytes (Kim et al., 2004). Despite lack of an oPPP in the apicoplast (Ralph et al., 2004), Plasmodium falciparum has retained its MEP, most likely because it lacks a cytosolic MVA (Imlay & Odom, 2014), an alternate source of isoprenoid production. Taken together, whether loss of photosynthesis leads to loss of the MEP likely depends on several interrelated factors, including whether the functionally overlapping MVA and MEP pathways (Liao et al., 2016) were both present in the photosynthetic ancestor and whether there is a dedicated plastid oPPP. Given the challenges of shedding the plastid entirely, the limited number of requisite supply lines to plastid‐localized biosynthetic pathways is likely an important constraint on loss of photosynthesis in groups without a plastid oPPP. In the absence of both PSI and an oPPP, the ease with which nonphotosynthetic diatoms meet their NADPH demands, either by eliminating dispensable pathways and increasing flux of plastid NADPH through a limited number of known pathways (Fig. 4), or whether they possess novel mechanisms for provisioning NADPH in their plastids, remains unclear.

Conclusions

Since their origin some 200 Myr ago, diatoms have become an integral part of marine and freshwater ecosystems worldwide. Photosynthesis by diatoms accounts for 20% of global primary production, making them cornerstones of the ‘biological pump’ that fixes and then buries atmospheric carbon on the seafloor. We sequenced the genome of a nonphotosynthetic diatom to understand how metabolism has been restructured in heterotrophic diatoms and, more broadly, how core aspects of diatom metabolism manifest at macroevolutionary scales, such as the frequency of transition between trophic modes.

Metabolic and genomic changes associated with the switch to heterotrophy in Nitzschia Nitz4 were cellular‐wide and included losses of photosynthesis‐related genes from the nuclear and plastid genomes, loss of the plastid MEP pathway, and remodeling of an ancient and conserved mode of mitochondrial glycolysis – a switch that may help compensate for the loss of autotrophic sources of ATP. Lack of a dedicated plastid oPPP is an ancestral feature of diatoms. Considering that groups which have lost photosynthesis more frequently tend to have a plastid oPPP, the comparative rarity of loss of photosynthesis in diatoms may reflect the fewer avenues available to diatoms to compensate for the loss of photosynthesis‐derived NADPH.

The presence of a β‐ketoadipate pathway underscores the genomic complexity of diatoms and the ability of genome sequencing to provide a first glimpse into the metabolism of marine microbes. As for other diatoms, we could not ascribe functions to many of the genes in Nitzschia Nitz4, potentially masking important aspects of its metabolism and highlighting a persistent challenge in comparative genomics.

Author contributions

AJA, JAL, AO and WRR designed the research. AJA, AO, ECR and WRR performed the research. AJA, JAL, AO and WRR wrote the article with input from all co‐authors.

Fig. S1 Contrasting inferences of gene family expansion and contraction in nonphotosynthetic diatoms based on analyses of protein‐coding genes from genomes only or genomes and transcriptomes.

Fig. S2 Light and scanning electron micrographs of Nitzschia sp. strain Nitz4.

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Methods S1 Genome assembly, contaminant removal, annotation, and analysis of heterozygosity in the nuclear genome of Nitzschia sp. Nitz4.

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Table S1 Plastid genome characteristics of five phylogenetically diverse diatoms.

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Table S2 Genome assembly characteristics for Nitzschia sp. Nitz4 and other diatoms.

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Table S3 Summary of variant calling for the nuclear genome of Nitzschia sp. Nitz4.

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Table S4 Annotation and localization of carbon metabolism genes in the genome of Nitzschia sp. Nitz4.

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Table S5 Annotation of carbon transporters in the genome of Nitzschia sp. Nitz4.

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Table S6 Annotation of carbohydrate‐active enzymes in the genome of Nitzschia sp. Nitz4.

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Table S7 Annotation information for genes in the β‐ketoadipate pathway of Nitzschia sp. Nitz4.

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Table S8 β‐Ketoadipate pathway genes in the sequenced genomes and transcriptomes of photosynthetic and nonphotosynthetic diatoms.

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