Phylogenomic placement, description, and mitochondrial genome of a novel freshwater goniomonad (Cryptista) from Arizona: Naiadagoniomonas ponderosa n. gen. n. sp.

Introduction

The Cryptista is a clade comprising the Cryptophyta along with poorly studied lineages of heterotrophic flagellates including the Katablepharida, the Goniomonadea, and the enigmatic genus Palpitomonas, ^1–6 as well as some environmental clades that include the described species Hemiarma marina.⁷ The three heterotrophic lineages of cryptists are inferred to be ancestrally heterotrophic, suggesting that cryptophytes independently acquired a single plastid derived from a red alga via secondary endosymbiosis.⁸ Goniomonads branch immediately sister to cryptophytes in phylogenomic analyses, so insights into the characters of goniomonads might help to understand the traits of the ancestral pre-plastid cryptophyte. The recently established marine goniomonad Neptunogoniomonas avonlea (Goniomonas avonlea originally) is currently the only representative of the group that has its genome sequenced.⁴

Goniomonads consisted of only a single genus (e.g.,⁹), until it was split into nine genera,¹⁰ and before a tenth genus was described in 2025.¹¹ These recent taxonomic advances leave us with a total of three described freshwater genera and another seven from marine environments. In phylogenetic trees inferred from 18S small subunit ribosomal DNA (SSU) sequences, there is a striking divergence between a freshwater clade and all other (marine) goniomonads.^3,11 This divergence suggests that there may have been only a single transition to freshwater in this lineage³ and that there may be substantial diversity of freshwater goniomonads remaining to be discovered.^10–12

Here we describe a new freshwater goniomonad, which we name Naiadagoniomonas ponderosa n. gen. n. sp. We used differential interference contrast (DIC) light microscopy and scanning electron microscopy (SEM) to show that it has a typical goniomonad morphology. We sequenced the 18S SSU rDNA gene from this cell, and it branched within the freshwater clade, sister to the genus Limnogoniomonas. We isolated single cells from the culture and generated a single-cell amplified genome (SAG) using whole genome amplification (WGA). The resulting N. ponderosa SAG has a high genome completeness score. Using the SAG data, we reconstructed a phylogenomic tree which shows Naiadagoniomonas ponderosa branching sister to N. avonlea with full support. In addition, we recovered a complete circular-mapping mitochondrial genome of this species. Its mitochondrial genome has a similar coding content to the previously published genome of N. avonlea but contained genes encoding subunits of the twin arginine translocase (Tat), further inviting questions about the diversity of mitochondrial genomes in this group.

Materials and methods

Results and discussion

Naiadagoniomonas ponderosa cells were observed using DIC on a Nikon Eclipse Ti microscope. The observed cells exhibited a characteristic goniomonad morphology,^32,33 with a flat oval-shape of length 6.94 μm ± 1.26 μm and width 4.60 μm ± 0.78 μm. Cells had two anterior flagella, each ~ 3.21 μm ± 0.46 μm long (0.46x cell length) and a band of visible ejectisomes near their anterior ( Figure 1A and Extended Data Video S1). Cells not attached to the surface of the cover slip swim and turn very quickly (see Extended Data Video S1, cells move into and out of field of view). Additionally, cells were observed spinning in place on the surface of the coverslip (see Extended Data Video S2).

Figure 1. Naiadagoniomonas ponderosa branches sister to Limnogoniomonas and has typical goniomonad characteristics.

A. Differential interference contrast (DIC) micrograph showing ejectisomes (arrowhead). Scale bar = 10 μm. B. Scanning electron micrograph (SEM) of the left side of a cell showing the periplast plate pattern characteristic of this species. Scale bar = 2 μm. C. SSU rDNA phylogeny of the Cryptista showing the position of Naiadagoniomonas ponderosa. This phylogenetic tree was estimated under a maximum-likelihood framework under GTR + Gamma model and 1000 nonparametric bootstrap replicates, rooted on the CRY-1 clade as an outgroup. Bootstrap values under 75 are not shown and nodes with > 98 support are marked by a black circle.

Goniomonad periplast plate patterns have been used to differentiate marine goniomonad species from one another in tandem with molecular phylogenetic analyses.⁹ Scanning electron micrographs reveal three periplast plates on the left side of N. ponderosa cells ( Figure 1B), similar to the plate patterns observed in some marine goniomonad genera (i.e., Marigoniomonas) rather than the described freshwater species.¹⁰ Additional studies investigating the ultrastructure of freshwater goniomonads using SEM is necessary to compare periplast plate patterns across related species.

The N. ponderosa filtered SAG assembly constituted a total length of 256 Mbp, 170,369 contigs with an N50 of 14 kbp. To assess the level of bacterial contamination of our SAG, we used blobtools, which showed a large degree of bacterial contamination before filtering (Extended Data Figure S1A). Because our filtered SAG assembly has only a single peak that hits eukaryotes, we believe the SAG has little if any eukaryotic contamination (Extended Data Figure S1B). Furthermore, when using the Neptunogoniomonas avonlea SSU as a BLASTn query into our SAG, only a the corresponding SSU from N. ponderosa was detected, suggesting that no other eukaryotic genome was amplified. As a final check for eukaryotic contamination, we looked for contaminating mitochondrial genomes. Because eukaryotic SAGs tend to amplify mitochondrial genomes, we used the DNA sequence of the cob gene from the N. avonlea mitochondrial genome as a BLASTn query into a database containing our SAG assembly. Though this gene sequence readily hits many mitochondrial gene sequences in the NCBI nucleotide database, apart from the N. ponderosa mitochondrial genome (see below), the search recovered only a single bacterial contig. We therefore conclude that the SAG has relatively little eukaryotic contamination. The N. ponderosa SAG achieved a remarkably high BUSCO score (71.4% complete and 11.4% fragmented) compared to other SAGs (often only between 0 and 10% (see e.g.³⁴). The published genome of N. avonlea contained more fragmented BUSCO markers but similar overall score (69% complete and 20% fragmented⁴). Though it is still possible that the SAG is contaminated with an unknown eukaryote, inspection of single gene trees within the PhyloFisher pipeline (see methods) suggest that most of the eukaryotic signal in this SAG is from N. ponderosa.

Next, we extracted the SSU rDNA sequence from our SAG assembly and reconstructed a single-gene phylogeny demonstrating that N. ponderosa branches within the freshwater clade of goniomonads, sister to the genus Limnogoniomonas, along with some environmental sequences ( Figure 1B). To confirm the placement of N. ponderosa in the tree of eukaryotes, we performed a phylogenomic analysis using the PhyloFisher pipeline.²⁸ As expected, N. ponderosa branches sister to the only other goniomonad in the dataset, N. avonlea, with full support ( Figure 2). The clade comprising Cryptophyta + goniomonads was also recovered with full support ( Figure 2).

Figure 2. Naiadagoniomonas ponderosa branches sister to Neptunogoniomonas avonlea in multigene phylogeny.

The PhyloFisher pipeline was used to estimate the phylogeny of eukaryotes using Archaeplastida as an outgroup resulting in a data matrix of 48 taxa and 103 genes (30,846 total amino acid sites). The Maximum Likelihood phylogeny was estimated using the LG+C10+F+G4 model with 1000 Ultrafast bootstraps (UFB) and 200 non-parametric bootstraps LG+C10+F+G4+posterior mean site frequency (PMSF). Naiadagoniomonas ponderosa is marked in red and bold font. Nodes with full support are marked with a black circle. Percentages of sites occupied per taxa is represented by orange bars.

In the N. ponderosa SAG, we identified a single 45,356 bp contig (30.6% GC content) representing its complete circular-mapping mitochondrial genome. The mitochondrial genome of N. ponderosa was similar in gene content compared to N. avonlea but lacked rpl10 and contained rps2 ( Figure 3). It additionally contained two protein-coding genes for the mitochondrial-encoded components of the bacteria-derived twin-arginine translocase (Tat), tatA and tatC. Though introns were identified in N. avonlea in the atp8 and cox1 genes, we identified introns in the nad2 and cox1 genes. The cox1 introns appear unrelated between the two species suggesting independent intron invasions. While rps2 is encoded in the mitochondrial genomes of katablepharids, Hemiarma marina from the CRY-1 clade,³⁵ and cryptophytes, but not in Palpitomonas^35,36 (see Figure 3). rpl10 is sporadically retained in various cryptists, though an inability to identify this gene in N. ponderosa could represent divergence beyond recognition. Meanwhile, tatA and tatC are absent in katablepharids but present in all other cryptists,^35,37,38 except N. avonlea. The function of TatA and TatC in eukaryotes remains mysterious, and it is unclear why some eukaryotes retain the genes while others do not.³⁹

Figure 3. The mitochondrial genome of Naiadagoniomonas ponderosa encodes a different set of proteins compared to that of Neptunogoniomonas avonlea.

Mitochondrial genome coding contents were predicted using MFannot or mined from published literature. A schematic representation of the Cryptista phylogeny is on the left. Filled boxes represent the presence of mitochondria-encoded genes with the goniomonad genes shaded in darker blue. Abbreviations: CRYP=Cryptomonada; GONI=Goniomonadea; C1=CRY-1 clade; KA=Kathablepharidacea; PA=Palpitomonadidae.

Conclusions

Here, we describe a new genus and species of freshwater goniomonad Naiadagoniomonas ponderosa. Our data suggest that freshwater goniomonads have been largely overlooked and several undescribed lineages likely inhabit various freshwater habitats. We produced a high-quality SAG with high genome completeness and show that N. ponderosa branches sister to Limnogoniomonas in a single-gene tree inferred from SSU rDNA sequences and with the sole goniomonad representative Neptunogoniomonas avonlea in a phylogenomic analysis. Finally, we describe its complete mitochondrial genome and discuss differences within the cryptist clade. In conclusion, our data provide the basis for future comparative genomic investigations of goniomonads that will help to explain how goniomonads have evolved to occupy different niches in both freshwater and marine habitats.

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