Cryptomonad Nucleomorph Research Articles

The cryptomonad nucleomorph.

The cryptomonad nucleomorph is a vestigial nucleus of a eukaryotic red alga engulfed by a phagotrophic protist and retained as a photosynthetic endosymbiont. This review recounts the initial discovery and subsequent characterisation of the cryptomonad nucleomorph focusing on the key role of Peter Sitte and his protégés in our understanding of secondary endosymbiosis to create complex plastids, one of the major transition events in the evolution of life on Earth.

Complete Nucleomorph Genome Sequence of the Nonphotosynthetic Alga Cryptomonas paramecium Reveals a Core Nucleomorph Gene Set

Nucleomorphs are the remnant nuclei of algal endosymbionts that were engulfed by nonphotosynthetic host eukaryotes. These peculiar organelles are found in cryptomonad and chlorarachniophyte algae, where they evolved from red and green algal endosymbionts, respectively. Despite their independent origins, cryptomonad and chlorarachniophyte nucleomorph genomes are similar in size and structure: they are both <1 million base pairs in size (the smallest nuclear genomes known), comprised three chromosomes, and possess subtelomeric ribosomal DNA operons. Here, we report the complete sequence of one of the smallest cryptomonad nucleomorph genomes known, that of the secondarily nonphotosynthetic cryptomonad Cryptomonas paramecium. The genome is 486 kbp in size and contains 518 predicted genes, 466 of which are protein coding. Although C. paramecium lacks photosynthetic ability, its nucleomorph genome still encodes 18 plastid-associated proteins. More than 90% of the “conserved” protein genes in C. paramecium (i.e., those with clear homologs in other eukaryotes) are also present in the nucleomorph genomes of the cryptomonads Guillardia theta and Hemiselmis andersenii. In contrast, 143 of 466 predicted C. paramecium proteins (30.7%) showed no obvious similarity to proteins encoded in any other genome, including G. theta and H. andersenii. Significantly, however, many of these “nucleomorph ORFans” are conserved in position and size between the three genomes, suggesting that they are in fact homologous to one another. Finally, our analyses reveal an unexpected degree of overlap in the genes present in the independently evolved chlorarachniophyte and cryptomonad nucleomorph genomes: ∼80% of a set of 120 conserved nucleomorph genes in the chlorarachniophyte Bigelowiella natans were also present in all three cryptomonad nucleomorph genomes. This result suggests that similar reductive processes have taken place in unrelated lineages of nucleomorph-containing algae.

Actin Gene Family Dynamics in Cryptomonads and Red Algae

Here we present evidence for a complex evolutionary history of actin genes in red algae and cryptomonads, a group that acquired photosynthesis secondarily through the engulfment of a red algal endosymbiont. Four actin genes were found in the nuclear genome of the cryptomonad, Guillardia theta, and in the genome of the red alga, Galdieria sulphuraria, a member of the Cyanidiophytina. Phylogenetic analyses reveal that the both organisms possess two distinct sequence types, designated "type-1" and "type-2." A weak but consistent phylogenetic affinity between the cryptomonad type-2 sequences and the type-2 sequences of G. sulphuraria and red algae belonging to the Rhodophytina was observed. This is consistent with the possibility that the cryptomonad type-2 sequences are derived from the red algal endosymbiont that gave rise to the cryptomonad nucleomorph and plastid. Red algae as a whole possess two very different actin sequence types, with G. sulphuraria being the only organism thus far known to possess both. The common ancestor of Rhodophytina and Cyanidiophytina may have had two actin genes, with differential loss explaining the distribution of these genes in modern-day groups. Our study provides new insight into the evolution and divergence of actin genes in cryptomonads and red algae, and in doing so underscores the challenges associated with heterogeneity in actin sequence evolution and ortholog/paralog detection.

Novel Nucleomorph Genome Architecture in the Cryptomonad Genus Hemiselmis

Cryptomonads are ubiquitous aquatic unicellular eukaryotes that acquired photosynthesis through the uptake and retention of a red algal endosymbiont. The nuclear genome of the red alga persists in a highly reduced form termed a nucleomorph. The nucleomorph genome of the model cryptomonad Guillardia theta has been completely sequenced and is a mere 551 kilobases (kb) in size, spread over three chromosomes. The presence of three chromosomes appears to be a universal characteristic of nucleomorph genomes in cryptomonad algae as well as in the chlorarachniophytes, an unrelated algal lineage with a nucleomorph and plastid genome derived from a green algal endosymbiont. Another feature of nucleomorph genomes in all cryptomonads and chlorarachniophytes examined thus far is the presence of subtelomeric ribosomal DNA (rDNA) repeats at the ends of each chromosome. Here we describe the first exception to this canonical nucleomorph genome architecture in the cryptomonad Hemiselmis rufescens CCMP644. Using pulsed-field gel electrophoresis (PFGE), we estimate the size of the H. rufescens nucleomorph genome to be approximately 580 kb, slightly larger than the G. theta genome. Unlike the situation in G. theta and all other known cryptomonads, sub-telomeric repeats of the rDNA cistron appear to be absent on both ends of the second largest chromosome in H. rufescens and two other members of this genus. Southern hybridizations using a variety of nucleomorph protein gene probes against PFGE-separated H. rufescens chromosomes indicate that recombination has been a major factor in shaping the karyotype and genomic structure of cryptomonad nucleomorphs.

Insight into the Diversity and Evolution of the Cryptomonad Nucleomorph Genome

Insight into the Diversity and Evolution of the Cryptomonad Nucleomorph Genome

Complete nucleotide sequence of the chlorarachniophyte nucleomorph: Nature’s smallest nucleus

The introduction of plastids into different heterotrophic protists created lineages of algae that diversified explosively, proliferated in marine and freshwater environments, and radically altered the biosphere. The origins of these secondary plastids are usually inferred from the presence of additional plastid membranes. However, two examples provide unique snapshots of secondary-endosymbiosis-in-action, because they retain a vestige of the endosymbiont nucleus known as the nucleomorph. These are chlorarachniophytes and cryptomonads, which acquired their plastids from a green and red alga respectively. To allow comparisons between them, we have sequenced the nucleomorph genome from the chlorarachniophyte Bigelowiella natans: at a mere 373,000 bp and with only 331 genes, the smallest nuclear genome known and a model for extreme reduction. The genome is eukaryotic in nature, with three linear chromosomes containing densely packed genes with numerous overlaps. The genome is replete with 852 introns, but these are the smallest introns known, being only 18, 19, 20, or 21 nt in length. These pygmy introns are shown to be miniaturized versions of normal-sized introns present in the endosymbiont at the time of capture. Seventeen nucleomorph genes encode proteins that function in the plastid. The other nucleomorph genes are housekeeping entities, presumably underpinning maintenance and expression of these plastid proteins. Chlorarachniophyte plastids are thus serviced by three different genomes (plastid, nucleomorph, and host nucleus) requiring remarkable coordination and targeting. Although originating by two independent endosymbioses, chlorarachniophyte and cryptomonad nucleomorph genomes have converged upon remarkably similar architectures but differ in many molecular details that reflect two distinct trajectories to hypercompaction and reduction.

Insight into the Diversity and Evolution of the Cryptomonad Nucleomorph Genome

The cryptomonads are an enigmatic group of marine and freshwater unicellular algae that acquired their plastids through the engulfment and retention of a eukaryotic ("secondary") endosymbiont. Together with the chlorarachniophyte algae, the cryptomonads are unusual in that they have retained the nucleus of their endosymbiont in a miniaturized form called a nucleomorph. The nucleomorph genome of the cryptomonad Guillardia theta has been completely sequenced and with only three chromosomes and a total size of 551 kb, is a model of nuclear genome compaction. Using this genome as a reference, we have investigated the structure and content of nucleomorph genomes in a wide range of cryptomonad algae. In this study, we have sequenced nine new cryptomonad nucleomorph 18S ribosomal DNA (rDNA) genes and four heat shock protein 90 (hsp90) gene fragments, and using pulsed-field gel electrophoresis and Southern hybridizations, have obtained nucleomorph genome size estimates for nine different species. We also used long-range polymerase chain reaction to obtain nucleomorph genomic fragments from Hanusia phi CCMP325 and Proteomonas sulcata CCMP704 that are syntenic with the subtelomeric region of nucleomorph chromosome I in G. theta. Our results indicate that (1) the presence of three chromosomes is a common feature of the nucleomorph genomes of these organisms, (2) nucleomorph genome size varies dramatically in the cryptomonads examined, (3) unidentified cryptomonad species CCMP1178 has the largest nucleomorph genome identified to date at approximately 845 kb, (4) nucleomorph genome size reductions appear to have occurred multiple times independently during cryptomonad evolution, (5) the relative positions of the 18S rDNA, ubc4, and hsp90 genes are conserved in three different cryptomonad genera, and (6) interchromosomal recombination appears to be rapidly changing the size and sequence of a repetitive subtelomeric region of the nucleomorph genome between the 18S rDNA and ubc4 loci. These results provide a glimpse into the genetic diversity of nucleomorph genomes in cryptomonads and set the stage for more comprehensive sequence-based studies in closely and distantly related taxa.

The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria-containing eukaryotes and an alternative hypothesis on the origin of plastids.

Red algae are one of the main photosynthetic eukaryotic lineages and are characterized by primitive features, such as a lack of flagella and the presence of phycobiliproteins in the chloroplast. Recent molecular phylogenetic studies using nuclear gene sequences suggest two conflicting hypotheses (monophyly versus non-monophyly) regarding the relationships between red algae and green plants. Although kingdom-level phylogenetic analyses using multiple nuclear genes from a wide-range of eukaryotic lineages were very recently carried out, they used highly divergent gene sequences of the cryptomonad nucleomorph (as the red algal taxon) or incomplete red algal gene sequences. In addition, previous eukaryotic phylogenies based on nuclear genes generally included very distant archaebacterial sequences (designated as the outgroup) and/or amitochondrial organisms, which may carry unusual gene substitutions due to parasitism or the absence of mitochondria. Here, we carried out phylogenetic analyses of various lineages of mitochondria-containing eukaryotic organisms using nuclear multigene sequences, including the complete sequences from the primitive red alga Cyanidioschyzon merolae. Amino acid sequence data for two concatenated paralogous genes (alpha- and beta-tubulin) from mitochondria-containing organisms robustly resolved the basal position of the cellular slime molds, which were designated as the outgroup in our phylogenetic analyses. Phylogenetic analyses of 53 operational taxonomic units (OTUs) based on a 1525-amino-acid sequence of four concatenated nuclear genes (actin, elongation factor-1alpha, alpha-tubulin, and beta-tubulin) reliably resolved the phylogeny only in the maximum parsimonious (MP) analysis, which indicated the presence of two large robust monophyletic groups (Groups A and B) and the basal eukaryotic lineages (red algae, true slime molds, and amoebae). Group A corresponded to the Opisthokonta (Metazoa and Fungi), whereas Group B included various primary and secondary plastid-containing lineages (green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, and Heterolobosea. The red algae represented the sister lineage to Group B. Using 34 OTUs for which essentially the entire amino acid sequences of the four genes are known, MP, distance, quartet puzzling, and two types of maximum likelihood (ML) calculations all robustly resolved the monophyly of Group B, as well as the basal position of red algae within eukaryotic organisms. In addition, phylogenetic analyses of a concatenated 4639-amino-acid sequence for 12 nuclear genes (excluding the EF-2 gene) of 12 mitochondria-containing OTUs (including C. merolae) resolved a robust non-sister relationship between green plants and red algae within a robust monophyletic group composed of red algae and the eukaryotic organisms belonging to Group B. A new scenario for the origin and evolution of plastids is suggested, based on the basal phylogenetic position of the red algae within the large clade (Group B plus red algae). The primary plastid endosymbiosis likely occurred once in the common ancestor of this large clade, and the primary plastids were subsequently lost in the ancestor(s) of the Discicristata (euglenoids, Kinetoplastida, and Heterolobosea), Heterokontophyta, and Alveolata (apicomplexans and Ciliophora). In addition, a new concept of "Plantae" is proposed for phototrophic and nonphototrophic organisms belonging to Group B and red algae, on the basis of the common history of the primary plastid endosymbiosis. The Plantae include primary plastid-containing phototrophs and nonphototrophic eukaryotes that possibly contain genes of cyanobacterial origin acquired in the primary endosymbiosis.

Molecular chaperones encoded by a reduced nucleus: the cryptomonad nucleomorph.

Molecular chaperones mediate the correct folding of nascent or denatured proteins and are found in both the organelles and cytoplasm of eukaryotic cells. Cryptomonad algae are unusual in possessing an extra cytoplasmic compartment (the periplastid space), the result of having engulfed and retained a photosynthetic eukaryote. Within the periplastid space is a diminutive nucleus (the nucleomorph) that encodes mostly genes for its own expression as well as a few needed by the plastid. Two plastid-encoded chaperones (GroEL and DnaK) and a nucleomorph-encoded chaperone (Cpn60) have been reported from the cryptomonad, Guillardia theta. Here we analyse G. theta nucleomorph genes for members of the cytosolic HSP70 and HSP90 families of molecular chaperones, a heat shock transcription factor (HSF), and all eight subunits of the group II chaperonin, CCT. These are presumably all active in the periplastid space, assisting in the maturation of polypeptides required by the cell; we propose a central role for them also in the structure and assembly of a putative relict mitotic apparatus. Curiously, none of the genes for co-chaperones of HSP70, HSP90, or CCT have been detected in the nucleomorph genome; they are either not needed or are encoded in the host nuclear genome and targeted back into the periplastid space. Endoplasmic reticulum (ER) homologs of HSP70 and HSP90 are also not present. Striking differences in the degree of conservation of the various nucleomorph-encoded molecular chaperones were observed. While the G. theta HSP70 and HSP90 homologs are well conserved, each of the eight CCT subunits (alpha, beta, gamma, delta, epsilon, eta, theta, and zeta) is remarkably divergent. Such differences are likely evidence for reduced/different functional constraints on the various molecular chaperones functioning in the periplastid space.

Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph.

Cells of several major algal groups are evolutionary chimeras of two radically different eukaryotic cells. Most of these "cells within cells" lost the nucleus of the former algal endosymbiont. But after hundreds of millions of years cryptomonads still retain the nucleus of their former red algal endosymbiont as a tiny relict organelle, the nucleomorph, which has three minute linear chromosomes, but their function and the nature of their ends have been unclear. We report extensive cryptomonad nucleomorph sequences (68.5 kb), from one end of each of the three chromosomes of Guillardia theta. Telomeres of the nucleomorph chromosomes differ dramatically from those of other eukaryotes, being repeats of the 23-mer sequence (AG)(7)AAG(6)A, not a typical hexamer (commonly TTAGGG). The subterminal regions comprising the rRNA cistrons and one protein-coding gene are exactly repeated at all three chromosome ends. Gene density (one per 0.8 kb) is the highest for any cellular genome. None of the 38 protein-coding genes has spliceosomal introns, in marked contrast to the chlorarachniophyte nucleomorph. Most identified nucleomorph genes are for gene expression or protein degradation; histone, tubulin, and putatively centrosomal ranbpm genes are probably important for chromosome segregation. No genes for primary or secondary metabolism have been found. Two of the three tRNA genes have introns, one in a hitherto undescribed location. Intergenic regions are exceptionally short; three genes transcribed by two different RNA polymerases overlap their neighbors. The reported sequences encode two essential chloroplast proteins, FtsZ and rubredoxin, thus explaining why cryptomonad nucleomorphs persist.

The Origin of Red Algae and Cryptomonad Nucleomorphs: A Comparative Phylogeny Based on Small and Large Subunit rRNA Sequences ofPalmaria palmata, Gracilaria verrucosa,and theGuillardia thetaNucleomorph

The complete large subunit rRNA sequences from the red algaePalmaria palmataandGracilaria verrucosa,and from the nucleomorph of the cryptomonadGuillardia theta,were determined in order to assess their phylogenetic relationships relative to each other and to other eukaryotes. Neighbor-joining, maximum-parsimony, and maximum-likelihood trees were constructed on the basis of small subunit rRNA, large subunit rRNA, and a combination of both molecules. Our results support the hypothesis that the cryptomonad plastid is derived from a primitive red alga, in that an ancient common ancestor of rhodophytes and cryptomonad nucleomorphs is indicated. This cluster shows some affinity with chlorobionts, which could point to a monophyletic origin of green and red plastids. However, the exact branching order of the crown eukaryotes remains uncertain and further research is required.

Cryptomonad nuclear and nucleomorph 18S rRNA phylogeny

Nuclear and nucleomorph 18S ribosomal RNA genes from six cryptomonads were amplified by the polymerase chain reaction and sequenced. Phylogenetic trees were constructed by distance, parsimony, and maximum likelihood methods for all available cryptomonad nuclear and nucleomorph 18S rRNA sequences. Nuclear and nucleomorph trees are largely congruent and clearly disprove the idea of polyphyletic origins for cryptomonad chloroplasts. Both show the leucoplast-containing Chilomonas as the sister to all photosynthetic cryptomonads. Using 11 cryptomonad nucleomorph sequences gives more convincing evidence than before that cryptomonad nucleomorphs originated from a red alga and are not specifically related to Chlorarachnion nucleomorphs. Both trees show as a clade the genera with nucleomorphs embedded in a chloroplast-envelope invagination into the pyrenoid (Storeatula, Rhinomonas, Rhodomonas). This monophyly of embedded nucleomorphs supports the recent creation of the order Pyrenomonadales for such cryptomonads. Cryptomonads ancestrally having free nucleomorphs are much more diverse. Komma and Chroomonas, with the blue accessory pigment phycocyanin, form a clade, as do Guillardia and Cryptomonas Φ, both with the red pigment phycoerythrin. The nucleomorph trees strongly show the blue Chroomonas/Komma clade as sister to all red-pigmented genera, but nuclear sequences support this weakly, if at all, being sensitive to taxon sampling. Red and blue cryptomonads probably diverged early by differential pigment loss. Nuclear sequences provide no clear evidence for the nature of the host that engulfed the ancestral symbiont. Our nuclear trees using an extensive selection of outgroups, and recent evidence from chloroplast DNA, are consistent with but do not positively support the view that the closest relatives of Cryptista (i.e. Cryptophyceae plus Goniomonadea) are the Chromobiota (i.e. Haptophyta plus Heterokonta, the latter including heterokont algae—phylum Ochrophyta), and that Cryptista and Chromobiota are appropriately classified as subkingdoms of the kingdom Chromista. Maximum likelihood often groups Goniomonas with Chilomonas, suggesting that Goniomonas may have lost both nucleomorph and plastid and that the cryptist common ancestor was photosynthetic.

EVIDENCE THAT THE NUCLEOMORPHS OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYCEAE) ARE VESTIGIAL NUCLEI: MORPHOLOGY, DIVISION AND DNA‐DAPI FLUORESCENCE1

ABSTRACTChlorarachnion reptans Geitler shows affinities to both the Chlorophyceae and the chloroplast endoplasmic reticulum‐containing chromophyte algae in possessing chlorophyll b and chloroplasts which are limited by four membranes, respectively. In the periplastidal compartment surrounding each of the four to eight chloroplasts of a C. reptans cell are putative eukaryotic‐sized ribosomes, scattered tubules and vesicles, and a small double‐membrane‐limited nucleus‐like organelle named the nucleomorph. The nucleomorphs display 4′‐6‐diamidino‐2‐phenylindole (DAPI)fluorescence which is sensitive to DNase digestion, but not to treatment with RNase. The nucleomorphs also contain a fibrillogranular body which resembles a nucleolus. Nucleomorph division occurs by the sequential infolding of the inner and outer envelope membranes and subsequent constriction in two, with no involvement of microtubules. In all these characteristics, the nucleomorphs of C. reptans are similar to the cryptomonad nucleomorph which has been hypothesized to be the vestigial nucleus of an ancestral red alga which gave rise to the chloroplasts of the Cryptophyceae. The presence of chlorophyll b and the contents and morphology of C. reptans chloroplast compartments suggest a green algal origin for the chloroplasts of these cells. The discovery of a second organism with a DNA‐containing, nucleus‐like organelle in its chloroplast compartment lends strong support to the hypothesis that the chloroplasts of many algae have evolved from eukaryotic endosymbionts.