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. 2025 Feb 10;61(2):267–274. doi: 10.1111/jpy.13545

Three reference genomes for freshwater diatom ecology and evolution

Wade R Roberts 1,✉, Andrew J Alverson 1
PMCID: PMC12044402  PMID: 39930529

Abstract

Diatoms are an important component of marine and freshwater ecosystems. Although the majority of described diatom species live in freshwater systems, genome sequencing efforts have focused primarily on marine species. Genomic resources for freshwater species have the potential to improve our understanding of diatom ecology and evolution, particularly in the context of major environmental shifts. We used long‐ and short‐read sequencing platforms to assemble reference genomes for three freshwater diatom species, all in the order Thalalassiosirales, which are abundant in the plankton of oceans, lakes, reservoirs, and rivers worldwide. We targeted three species that cover the breadth of phylogenetic diversity in the cyclostephanoid clade of Thalassiosirales: Cyclostephanos tholiformis (JALLPB020000000), Discostella pseudostelligera (JALLBG020000000), and Praestephanos triporus (JALLAZ020000000). The reference genome for D. pseudostelligera was considerably smaller (39 Mb) than those of both P. triporus (73 Mb) and C. tholiformis (177 Mb). Long‐read sequencing allowed for the assembly of scaffold‐level genomes, including regions rich in repetitive DNA. Compared to short‐read assemblies, long‐read assemblies increased the contig N50 length as much as 37‐fold and reduced the number of contigs by more than 88%. Transcriptome‐guided annotation of the protein‐coding genes identified between 10,000 and 12,000 genes. This work provides further demonstration of the value of long‐read sequencing and provides novel genomic resources for understanding the ecology and evolution of freshwater diatoms.

Keywords: Cyclostephanos, diatoms, Discostella, long‐read sequencing, Praestephanos, salinity, Thalassiosirales

Abbreviations

AED

annotation edit distance

TSA

transcriptome shotgun assembly

WGS

whole genome shotgun

INTRODUCTION

Diatoms are ubiquitous across marine and freshwater ecosystems where they play key roles in global primary production and form the base of aquatic food webs (Armbrust, 2009; Smol & Stoermer, 2010). Approximately 70% of described diatom species exist in freshwaters (Nakov et al., 2019), yet most genome sequencing projects to date have focused on marine taxa. Available draft genomes of freshwater diatoms are useful for comparative genomics and gene identification but are often incomplete due to poor or missing coverage of repetitive DNA, which are the principal driver of genome size in diatoms (Galachyants et al., 2015; Maberly et al., 2021; Roberts et al., 2024). Long‐read sequencing technologies have made it possible to produce reference‐quality genomes that contain a full representation of an organism's DNA, including regulatory and repetitive regions. However, to date only two reference‐quality genomes have been published for freshwater diatoms, and both are in the pennate clade (Suzuki et al., 2022; Zepernick et al., 2022). Reference genomes create new possibilities for comparative genomic studies of individual species (Armbrust et al., 2004), populations (Pinseel et al., 2023), or clades (Roberts et al., 2024).

Thalassiosirales are a lineage of polar centric diatoms that are common and abundant in marine and freshwater phytoplankton communities. They have long been used as experimental models (Guillard & Ryther, 1962; Schultz, 1971; Thamatrakoln & Hildebrand, 2007), and one species, Cyclotella nana (formerly Thalassiosira pseudonana), was the first diatom with a sequenced genome (Armbrust et al., 2004), establishing it as an important model species (Poulsen & Kröger, 2023). Although genomic resources for Thalassiosirales are steadily increasing, most work has focused on marine and euryhaline species (Figure 1; Roberts et al., 2020, Liu et al., 2023, Liu & Chen, 2024a, 2024b). We previously assembled draft genomes using Illumina short‐read sequencing for three freshwater species: Cyclostephanos tholiformis, Discostella pseudostelligera, and Praestephanos triporus (Roberts et al., 2023). Although these assemblies contained most or all the expected genes, differences between the sequenced and estimated genome sizes revealed that genome coverage was incomplete due, most likely, to the misassembly of repetitive regions (Roberts et al., 2024). We have augmented previous assemblies with long‐read nanopore sequencing and report scaffold‐level, reference‐quality genome assemblies for these three freshwater diatoms.

FIGURE 1.

FIGURE 1

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Long‐read sequencing greatly improved genome assemblies for the freshwater diatoms Cyclostephanos tholiformis, Discostella pseudostelligera, and Praestephanos triporus. (a) Maximum likelihood phylogenetic analysis of Thalassiosirales reference genomes based on 100 concatenated single‐copy orthologs. All branches received maximum support based on 10,000 ultrafast bootstrap replicates. The scale bar represents the expected number of substitutions per site. (b) Increased length of the contig N50 length (in kilobases) and (c) reduction in the number of contigs between the long‐read (v2) genome assemblies versus the previous short‐read (v1) assemblies.

MATERIALS AND METHODS

Culturing, DNA extraction, and sequencing

Clonal cultures of Cyclostephanos tholiformis strain UTEX 3231 (AJA228‐03), Discostella pseudostelligera strain UTEX 3233 (AJA232‐27), and Praestephanos triporus strain AJA276‐08 were established from phytoplankton samples collected from three river systems in the southeastern United States (Table 1; Roberts et al., 2023). The cultures are available through the UTEX Culture Collection of Algae at the University of Texas at Austin. We extracted DNA using a modified CTAB protocol (Doyle & Doyle, 1987; Roberts et al., 2020) and constructed short‐read DNA libraries with the KAPA HyperPrep Kit (Roche) for 2 × 100 bp paired‐end sequencing on the Illumina HiSeq 4000 or NovaSeq 6000 platforms. Raw reads were quality‐filtered and trimmed of adapters with Ktrim (v1.5.1) (Sun, 2020), followed by error correction with BayesHammer (part of SPAdes 3.14.1) (Nikolenko et al., 2013).

TABLE 1.

General features of the genome assemblies.

Cyclostephanos tholiformis Discostella pseudostelligera Praestephanos triporus
Collection
Strain UTEX 3231 (AJA228‐03) UTEX 3233 (AJA232‐27) AJA276‐08
Locality Neosho River, Monkey Island, OK, USA Mississippi River, St. Louis, MO, USA Great Pee Dee River, Georgetown, SC, USA
Latitude, longitude 36.558683, −94.8384 38.622652, −90.183879 33.365217, −79.266267
Genome assembly
Estimated haploid genome size (Mb) 190.4 39.4 85.6
Assembly size (Mb) 177.4 39.1 72.8
Sequencing depth, Illumina 84× 132× 45×
Sequencing depth, ONT 21× 10× 10×
Number of scaffolds 914 316 1854
Contig N50 (kb) 260 194 51
L50 208 58 449
GC content (%) 47.6 47.2 49.9
Repetitive DNA (%) 74.2 21.5 47.1
Stramenopile BUSCO completeness of assembly (%) 94 97 92
Eukaryota BUSCO completeness of assembly (%) 71 75 68
NCBI WGS Accession JALLPB020000000 JALLBG020000000 JALLAZ020000000
Genome annotation
Number of protein‐coding genes 11,917 10,456 11,380
Average gene length (bp) 2089 2188 1915
Average exon length (bp) 708 861 694
Genes with annotation edit distance ≤0.5 (%) 98 98.4 96.2
Predicted proteins with Pfam or PANTHER domain (%) 70 71 67
BUSCO completeness of annotation (%) 94 95 88
OMArk completeness of annotation (%) 91.7 92.9 89.3
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Additional CTAB DNA extractions that contained sufficient quantities (>3 μg) of high molecular weight DNA were used for long‐read sequencing on the MinION platform (Oxford Nanopore Technologies, ONT). The quality and quantity of high molecular weight DNA was assessed using 0.8% agarose gel electrophoresis, Qubit 2.0 (dsDNA BR kit; Thermo Fisher Scientific), and Nanodrop 2000 (Thermo Fisher Scientific). Libraries were constructed with the Ligation Sequencing Kit (ONT) and sequenced on R9.4.1 flowcells (ONT). Raw MinION sequences were base‐called with ONT's Guppy software (‐‐trim_adapters ‐c dna_r.9.4.1_450bps_fast.cfg; v6.5.7); read quality statistics were calculated with NanoStat (v1.2.0; De Coster et al., 2018), and base‐called reads were self‐corrected with VeChat (v1.1.1; Luo et al., 2022).

Genome assembly

Default settings were used for all subsequent programs unless stated otherwise. Each stage of the assembly process was assessed for quality and completeness with QUAST (v5.0.0; Gurevich et al., 2013), BUSCO (stramenopiles_odb10 dataset; v5.1.3; Simão et al., 2015), and Merqury (v1.3; Rhie et al., 2020). Uncorrected long reads were assembled with metaFlye (‐‐meta ‐‐iterations 0; v2.9.1‐b1780; Kolmogorov et al., 2019, 2020) and corrected with Racon (‐m 8 ‐x ‐6 ‐g ‐8 ‐w 500; v1.4.16; Vaser et al., 2017) and Medaka (‐m r941_min_fast_g303; v1.2.3; https://github.com/nanoporetech/medaka). For Cyclostephanos tholiformis and Discostella pseudostelligera, the long‐read assemblies were polished five times using POLCA (part of MaSuRCA v.4.1.1; Zimin & Salzberg, 2020) and the short Illumina reads to fix single nucleotide and small indel errors. Next, the contigs were scaffolded and gap‐filled into longer contigs with the corrected long‐reads using SAMBA (‐d ont ‐m 3000 ‐f and ‐d ot ‐m 2000 ‐f; part of MaSuRCA v.4.1.1; Zimin & Salzberg, 2022). The scaffolds were then polished with JASPER (Guo et al., 2023). Following these steps, contaminant contigs belonging to bacteria were removed from the assemblies with EukRep (v0.6.6; West et al., 2018), and haplotigs were removed from the C. tholiformis assembly with Purge Haplotigs (Roach et al., 2018). For Praestephanos triporus, a hybrid assembly was performed using hybridSpades (‐‐nanopore ‐‐trusted‐contigs ‐k auto ‐‐cov‐cutoff off; v3.14.1; Antipov et al., 2016) that combined the short Illumina reads, long nanopore reads, and the corrected long‐read contigs. Following hybrid assembly, the short reads were used for scaffolding with SSPACE (v3.0; Boetzer et al., 2011), gap filling with GapCloser (v1.12; Luo et al., 2012), and polishing with POLCA. We then produced scaffolds with the corrected long reads using SAMBA and removed contaminant contigs from the assembly with EukRep. Finally, short contigs and remaining organellar contigs were removed from all three assemblies using BioKIT (remove_short_sequences; v0.0.9; Steenwyk et al., 2022).

Genome annotation and gene prediction

Repetitive sequences were identified with RepeatModeler2 (v.2.0.5; Flynn et al., 2020) and masked using RepeatMasker (‐s ‐a ‐gff ‐norna ‐xsmall; v.4.1.5; https://www.repeatmasker.org/). After masking, gene models were predicted using the MAKER2 pipeline (v2.31.10; Holt & Yandell, 2011), which integrates evidence from transcripts and proteins homologs with trained ab initio gene prediction models. For transcript evidence, we used previously generated de novo assembled transcriptomes from the three strains (Roberts et al., 2023). Predicted proteomes from Cyclotella cryptica CCMP332 v2 (Roberts et al., 2020), Cyclotella nana CCMP1335 v4 (Filloramo et al., 2021), Fragilariopsis cylindrus CCMP1102 v1 (Mock et al., 2017), and Phaeodactylum tricornutum CCAP1055/1 v3 (Rastogi et al., 2018) were used as protein homolog evidence for gene annotation. We ran MAKER first, using only the transcript and protein evidence to generate initial gene models. Subsequent rounds of MAKER included trained ab initio gene prediction models from Augustus (Stanke et al., 2008), GeneMark‐ES (Lomsadze et al., 2005), and SNAP (Korf, 2004). Augustus models were trained using the automated approach implemented in BUSCO with the eukaryota_odb10 dataset (‐‐augustus ‐‐long). GeneMark‐ES models were trained using a repeat soft‐masked version of the genome assembly. SNAP models were trained using the initial gene models predicted by MAKER from the alignments of proteins and transcripts to the genome assembly. MAKER gene models were assessed using annotation edit distance (AED; Holt & Yandell, 2011), recovery of conserved orthologs with BUSCO (stramenopiles_odb10 and eukaryota_odb10 datasets; v.5.1.3; Simão et al., 2015), and proteome completeness with OMArk (LUMA.h5 database; v.0.3.0; Nevers et al., 2024). Annotation edit distance scores were calculated for each predicted gene based on how congruent the gene model is with the input evidence (proteins and transcripts; Holt & Yandell, 2011). Annotations are considered excellent when 90% of gene models have AED ≤0.5 and more than 50% of the proteins have a recognizable domain (Campbell et al., 2014). Single‐exon gene overprediction can be a problem for poorly annotated genomes, so we also calculated the ratio of monoexonic:multiexonic genes using the output of gFACs (v.1.1.2; Caballero & Wegrzyn, 2019), as suggested by Vuruputoor et al. (2023).

To identify protein domains and gene ontology terms, we searched the predicted proteins against the Pfam (v.32.0; El‐Gebali et al., 2019) and PANTHER (v.14.1; Mi et al., 2019) databases using InterProScan (‐iprlookup ‐dp ‐goterms; v.5.36‐75.0; Jones et al., 2014). We also searched the proteins against the SwissProt database (release 2024_06) using NCBI BLASTP (‐evalue 1e‐10 ‐num_alignments 1 ‐seg yes ‐soft_masking true ‐lcase_masking ‐max_hsps 1; v.2.16.0; Camacho et al., 2009) and the UniProt Reference Proteomes database (release 2020_04) using Diamond BLASTP (‐‐evalue 1e‐10 ‐‐max‐target‐seqs 1 ‐‐sensitive ‐‐max‐hsps 1; v.2.1.9; Buchfink et al., 2015).

Genome size estimation

We estimated the haploid genome size of each strain using the kmer‐based approach implemented in GenomeScope (v.2.0; Ranallo‐Benavidez et al., 2020). Contaminant‐free short reads were prepared for each strain by aligning the Illumina reads to the final assembly using minimap2 (commands: ‐ax sr; v.2.10; Li, 2018), sorting the alignments by coordinate using samtools (v.1.10; Li et al., 2009), and exporting only the aligned and properly paired reads using bam2fastq (‐‐aligned ‐‐no‐unaligned ‐‐no‐filtered; https://github.com/jts/bam2fastq). K‐mers of length 31 were then counted and summarized into a histogram with Jellyfish (v.2.3.0). Finally, we provided the histogram to GenomeScope (‐k 31 ‐p 2) to estimate the haploid genome sizes.

Phylogenetic analysis

We used the 100 single‐copy orthologs from the BUSCO stramenopiles_odb10 dataset to estimate a phylogenetic tree. BUSCO orthologs were extracted from the three genome assemblies and combined with corresponding orthologs from the reference genomes of Cyclotella cryptica, Cyclotella nana, Skeletonema costatum CNS00243 v1 (Liu & Chen, 2024a), S. marinoi CNS00100 v1 (Liu et al., 2023), and S. tropicum CNS00166 v1 (Liu & Chen, 2024b). Individual ortholog alignments were generated with MAFFT (‐‐localpair ‐‐maxiterate 1000; v7.505; Katoh & Standley, 2013), trimmed with ClipKIT (‐m smart‐gap; v2.1.3; Steenwyk et al., 2020), and concatenated into a supermatrix with phyx (Brown et al., 2017). We estimated a maximum likelihood tree with IQ‐TREE (v2.2.0.3; Minh et al., 2020), partitioning the alignment by ortholog, identifying the best‐fit amino acid substitution model for each partition (‐m MFP ‐mset JTT, WAG, LG, Q.pfam), and performing 10,000 ultrafast bootstrap replicates (‐B 10000; Minh et al., 2013).

RESULTS AND DISCUSSION

We sequenced three libraries on the MinION platform and base‐called between roughly 308,000 and 594,000 reads per species that yielded 1–4 Gb of raw data (Appendix S1: Table S1). The N50 read lengths were 9.5–15.8 kb, and average Phred scores of reads across runs had approximately 92% accuracy (Table S1). Previous Illumina sequencing yielded 3–10 Gb of raw reads per species (Table S1).

We assembled long‐reads for Cyclostephanos tholiformis and Discostella pseudostelligera directly into draft genomes, but lower coverage of the Praestephanos triporus long‐reads necessitated a hybrid assembly that combined the long and short Illumina reads (Appendix S1: Table S2). As expected with nanopore‐based long‐read sequencing, the initial assemblies for C. tholiformis and D. pseudostelligera were highly contiguous (contig N50 >150 kb) but had low consensus quality values (QV) of ~17 (~98% accuracy). After multiple rounds of polishing with the higher accuracy Illumina reads, QV for the final assemblies for C. tholiformis and D. pseudostelligera increased to 32, consistent with ~99.9% accuracy. Since the P. triporus genome was assembled in a hybrid manner, there was a tradeoff between slightly smaller contigs (contig N50 of 50.8 kb) but higher accuracy (QV of 44, or 99.99% accuracy). Despite these different approaches, the final genome assemblies for each species were highly complete with Stramenopile BUSCO scores all >92% (Table 1; Table S2), which is standard for high‐quality diatom genome assemblies.

The inclusion of long nanopore reads dramatically improved previously published short‐read draft assemblies (Roberts et al., 2023). For each species, the contig N50 lengths increased 7‐fold (Praestephanos triporus), 13‐fold (Discostella pseudostelligera), and >37‐fold (Cyclostephanos tholiformis; Figure 1). These contig N50 values are similar to other diatom genomes sequenced on the nanopore sequencing platform (Audoor et al., 2024). Additionally, the inclusion of long‐read sequencing reduced the number of contigs for each species genome by >88% (Figure 1). Although chromosome‐level genome assemblies are becoming more common for diatoms (Filloramo et al., 2021; Liu & Chen, 2024a), our results demonstrate that long nanopore reads are sufficient to assemble complete or near‐complete diatom genomes into a few hundred contigs. Although these are not yet chromosome‐level assemblies, we detected telomeric sequence repeats (TTAGGG/CCCTAA; Fulnecková et al., 2013) at the beginning or end of five contigs in C. tholiformis, two in D. pseudostelligera, and five in P. triporus.

At more than 177 Mb in length, Cyclostephanos tholiformis had the largest genome assembly, while Praestephanos triporus was smaller at 73 Mb, and Discostella pseudostelligera was the smallest at 39 Mb (Table 1). These assembly sizes are consistent with the estimated haploid genome size for these species and strains (Table 1) and are also within the size range of other reference genomes published for the Thalassiosirales (Cyclotella nana, 33 Mb; Cyclotella cryptica, 171 Mb). The larger genomes contained proportionally more repetitive DNA (Table 1), further underscoring repeat content as the principal determinant of genome size in diatoms (Roberts et al., 2024). The large genome of C. tholiformis was composed of 74% repetitive DNA (Table 1), which is greater than the similarly sized Cyclotella cryptica genome (171 Mb) that had an estimated repetitive DNA content of 60% (Roberts et al., 2020). Unclassified repeats and long terminal retrotransposons were the largest contributors to the total repeat content in each genome (Appendix S1: Table S3). This trend is similar to that of other diatom genomes (Roberts et al., 2020) and confirms the need to understand how repeats and transposable elements contribute to diatom genomic diversity and evolution (Hermann et al., 2014).

Despite substantial differences in assembly lengths among Cyclostephanos tholiformis, Discostella pseudostelligera, and Praestephanos triporus, the three species contained similar numbers of predicted protein‐coding genes (Table 1). All three genomes had 10–12k genes, similar to the expected range of 10–20 k genes in other Thalassiosirales genomes (Roberts et al., 2023, 2024). The number of genes was similar between the new assemblies and older draft assemblies (Appendix S1: Table S4), showing the utility of short‐read‐based genome skimming for phylogenomics (Roberts et al., 2023, 2024). The high percentage (>96%) of gene models with AED ≤0.5 and the large percentage of predicted proteins with an identifiable protein domain (>67%) all suggested high‐quality annotations (Table 1). Additionally, the ratios of monoexonic:multiexonic genes were 0.65, 0.71, and 0.57 for C. tholiformis, D. pseudostelligera, and P. triporus, respectively. These values are lower than other recently published diatom genomes (e.g., Cyclotella nana, 0.89; Skeletonema marinoi, 1.19), suggesting that our gene models were not likely to suffer from single exon gene overpredictions. Putative functional annotations were identified for roughly 90% of gene models after searching the predicted proteins against the SwissProt, UniProt Reference Proteomes, Pfam, and PANTHER databases (Table S4).

Marine–freshwater transitions have spawned enormous diversity across the tree of life (Jamy et al., 2022), including for diatoms (Nakov et al., 2019). A complete understanding of how diatoms and other microeukaryotes repeatedly have crossed this and other steep environmental gradients will benefit from genetic studies of model species (Poulsen & Kröger, 2023) and cross‐species comparative genome studies. The scaffold‐level reference genomes and gene models for Cyclostephanos tholiformis, Discostella pseudostelligera, and Praestephanos triporus represent valuable new resources for studying how diatoms scale the “invisible wall” that separates marine and freshwater environments (Bilcke & Kamakura, 2023).

AUTHOR CONTRIBUTIONS

Wade R. Roberts: Conceptualization (equal); data curation (lead); formal analysis (lead); methodology (lead); validation (lead); visualization (lead); writing – original draft (equal); writing – review and editing (equal). Andrew J. Alverson: Conceptualization (equal); funding acquisition (lead); resources (lead); writing – original draft (equal); writing – review and editing (equal).

Supporting information

Appendix S1. Detailed results for sequencing output, genome assembly, repeat classification, and comparison to previous genome versions.

JPY-61-267-s001.xlsx (17.9KB, xlsx)

ACKNOWLEDGMENTS

We thank Elizabeth Ruck and Kala Downey for culture care and laboratory assistance. This work was supported by the National Science Foundation (DEB 1651087). This research used resources available through the Arkansas High Performance Computing Center, which is funded through multiple NSF grants and the Arkansas Economic Development Commission. The authors declare that they have no conflicts of interest.

Roberts, W. R. , & Alverson, A. J. (2025). Three reference genomes for freshwater diatom ecology and evolution. Journal of Phycology, 61, 267–274. 10.1111/jpy.13545

Editor: N. Poulsen

DATA AVAILABILITY STATEMENT

The Whole Genome Shotgun (WGS) projects are available from NCBI GenBank under accessions JALLPB020000000 (Cyclostephanos tholiformis), JALLBG020000000 (Discostella pseudostelligera), and JALLAZ020000000 (Praestephanos triporus). The Transcriptome Shotgun Assembly (TSA) projects are available from NCBI GenBank under the accessions GJWN00000000 (C. tholiformis), GKZL00000000 (D. pseudostelligera), and GKZM00000000 (P. triporus). The genome sequences, gene models, functional annotations, and code to reproduce Figure 1 have been deposited into Zenodo (https://doi.org/10.5281/zenodo.14628780).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1. Detailed results for sequencing output, genome assembly, repeat classification, and comparison to previous genome versions.

JPY-61-267-s001.xlsx (17.9KB, xlsx)

Data Availability Statement

The Whole Genome Shotgun (WGS) projects are available from NCBI GenBank under accessions JALLPB020000000 (Cyclostephanos tholiformis), JALLBG020000000 (Discostella pseudostelligera), and JALLAZ020000000 (Praestephanos triporus). The Transcriptome Shotgun Assembly (TSA) projects are available from NCBI GenBank under the accessions GJWN00000000 (C. tholiformis), GKZL00000000 (D. pseudostelligera), and GKZM00000000 (P. triporus). The genome sequences, gene models, functional annotations, and code to reproduce Figure 1 have been deposited into Zenodo (https://doi.org/10.5281/zenodo.14628780).

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