Centrohelida is still searching for a phylogenetic home: analyses of seven Raphidiophrys contractilis genes

By recent advance in evolutionary biology, the majority of eukaryotes are classified into six eukaryotic assemblages called as "supergroups". However, several eukaryotic groups show no clear evolutionary affinity to any of the six supergroups. Centrohelida, one of major heliozoan groups, are such an unresolved lineage. In this study, we newly determined the genes encoding translation elongation factor 2 (EF2), cytosolic heat shock protein 70 (HSP70), and cytosolic heat shock protein 90 (HSP90) from the centroheliozoan Raphidiophrys contractilis. The three Raphidiophrys genes were then combined with previously determined actin, alpha-tubulin, beta-tubulin, and SSU rRNA sequences to phylogenetically analyze the position of Centrohelida in global eukaryotic phylogeny. Although the multi-gene data sets examined in this study are the largest ones including the centroheliozoan sequences, the relationships between Centrohelida and the eukaryotic groups considered were unresolved. Our careful investigation revealed that the phylogenetic estimates were highly sensitive to genes included in the multi-gene alignment. The signal of SSU rRNA and that of alpha-tubulin appeared to conflict with one another: the former strongly prefers a monophyly of Diplomonadida (e.g., Giardia), Parabasalia (e.g., Trichomonas), Heterolobosea (e.g., Naegleria), and Euglenozoa (e.g., Trypanosoma), while the latter unites Diplomonadida, Parabasalia, Metazoa, and Fungi. In addition, EF2 robustly unites Rhodophyta and Viridiplantae, while the remaining genes considered in this study do not positively support the particular relationship. Thus, it is difficult to identify the phylogenetic relatives of Centrohelida in the present study, since strong (and some are conflicting) gene-specific "signals" are predominant in the current multi-gene data. We concluded that larger scale multi-gene phylogenies are necessary to elucidate the origin and evolution of Centrohelida.     

A new scenario of plastid evolution: plastid primary endosymbiosis before the divergence of the “Plantae,” emended

A recent hypothesis on the origin of eukaryotic phototrophs proposes that red algae, green plants (land plants plus green algae), and glaucophytes constitute the primary photosynthetic eukaryotes, whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis, whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary endosymbiotic events involving a phototrophic eukaryote and a host cell. However, the phylogenetic relationships among the three lineages of primary photosynthetic eukaryotes remained unresolved because previous nuclear multigene phylogenies used incomplete red algal gene sequences derived mainly from Porphyra (Rhodophyceae, one of the two lineages of the Rhodophyta), and lacked sequences from the Cyanidiophyceae (the other red algal lineage). Recently, the complete nuclear genome sequences from the red alga Cyanidioschyzon merolae 10D of the Cyanidiophyceae were determined. Using this genomic information, nuclear multigene phylogenetic analyses of various lineages of mitochondrion-containing eukaryotes were conducted. Since bacterial and amitochondrial eukaryotic genes present serious problems to eukaryotic phylogenies, basal eukaryotes were deduced based on the paralogous comparison of the concatenated - and -tubulin. The comparison demonstrated that cellular slime molds (Amoebozoa) represent the most basal position within the mitochondrion-containing organisms. With the cellular slime molds as the outgroup, phylogenetic analyses based on a 1,525-amino acid sequence of four concatenated nuclear genes [actin, elongation factor-1( EF-1), -tubulin, and -tubulin] resolved the presence of two large, robust monophyletic groups and the basal eukaryotic lineages (Amoebozoa). One of the two groups corresponded to the Opisthokonta (Metazoa and Fungi), whereas the other included various lineages containing primary and secondary plastids (red algae, green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, dinoflagellates, and Heterolobosea, for which the red algae represented the most basal lineage. Therefore, the plastid primary endosymbiosis likely occurred once in the common ancestor of the latter group, and the primary plastids were subsequently lost in the ancestor(s) of organisms within the group that now lacks primary plastids. A new concept of Plantae was proposed for phototrophic and nonphototrophic organisms belonging to this group on the basis of their common history of plastid primary endosymbiosis. This new scenario of plastid evolution is discussed here, and is compared with recent genome information and findings on the secondary endosymbiosis of the Euglena plastid.     

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

Abstract 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 (α- and β-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-1α, α-tubulin, and β-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.     

Phylogenetic analysis of the Glomeromycota by partial β-tubulin gene sequences

The 3’ end of the β-tubulin gene was amplified from 50 isolates of 45 species in Glomeromycota. The analyses included a representative selection of all families except Pacisporaceae and Geosiphonaceae. Phylogenetic analyses excluded three intron regions at the same relative positions in all species due to sequence and length polymorphisms. The β-tubulin gene phylogeny was similar to the 18S rRNA gene phylogeny at the family and species level, but it was not concordant at the order level. Species in Gigasporaceae and Glomeraceae grouped together but without statistical support. Paralogous sequences in Glomus species likely contributed to phylogenetic ambiguity. Trees generated using different fungal phyla as out-groups yielded a concordant topology. Family relationships within the Glomeromycota did not change regardless if the third codon position was included or excluded from the analysis. Multiple clones from three isolates of Scutellospora heterogama yielded divergent sequences. However, phylogenetic patterns suggested that only a single copy of the β-tubulin gene was present, with variation attributed to intraspecific sequence divergence.     

The testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa

Testate lobose amoebae (order Arcellinida Kent, 1880) are common in all aquatic and terrestrial habitats, yet they are one of the last higher taxa of unicellular eukaryotes that has not found its place in the tree of life. The morphological approach did not allow to ascertain the evolutionary origin of the group or to prove its monophyly. To solve these challenging problems, we analyzed partial small-subunit ribosomal RNA (SSU rRNA) genes of seven testate lobose amoebae from two out of the three suborders and seven out of the 13 families belonging to the Arcellinida. Our data support the monophyly of the order and clearly establish its position among Amoebozoa, as a sister-group to the clade comprising families Amoebidae and Hartmannellidae. Complete SSU rRNA gene sequences from two species and a partial actin sequence from one species confirm this position. Our phylogenetic analyses including representatives of all sequenced lineages of lobose amoebae suggest that a rigid test appeared only once during the evolution of the Amoebozoa, and allow reinterpretation of some morphological characters used in the systematics of Arcellinida.     

Phylogeny of Choanozoa,Apusozoa, and Other Protozoa and Early Eukaryote Megaevolution

Abstract The primary diversification of eukaryotes involved protozoa, especially zooflagellates—flagellate protozoa without plastids. Understanding the origins of the higher eukaryotic kingdoms (two purely heterotrophic, Animalia and Fungi, and two primarily photosynthetic, Plantae and Chromista) depends on clarifying evolutionary relationships among the phyla of the ancestral kingdom Protozoa. We therefore sequenced 18S rRNA genes from 10 strains from the protozoan phyla Choanozoa and Apusozoa. Eukaryote diversity is encompassed by three early-radiating, arguably monophyletic groups: Amoebozoa, opisthokonts, and bikonts. Our taxon-rich rRNA phylogeny for eukaryotes allowing for intersite rate variation strongly supports the opisthokont clade (animals, Choanozoa, Fungi). It agrees with the view that Choanozoa are sisters of or ancestral to animals and reveals a novel nonflagellate choanozoan lineage, Ministeriida, sister either to choanoflagellates, traditionally considered animal ancestors, or to animals. Maximum likelihood trees suggest that within animals Placozoa are derived from medusozoan Cnidaria (we therefore place Placozoa as a class within subphylum Medusozoa of the Cnidaria) and hexactinellid sponges evolved from demosponges. The bikont and amoebozoan radiations are both very ill resolved. Bikonts comprise the kingdoms Plantae and Chromista and three major protozoan groups: alveolates, excavates, and Rhizaria. Our analysis weakly suggests that Apusozoa, represented by Ancyromonas and the apusomonads (Apusomonas and the highly diverse and much more ancient genus Amastigomonas, from which it evolved), are not closely related to other Rhizaria and may be the most divergent bikont lineages. Although Ancyromonas and apusomonads appear deeply divergent in 18S rRNA trees, the trees neither refute nor support the monophyly of Apusozoa. The bikont phylum Cercozoa weakly but consistently appears as sister to Retaria (Foraminifera; Radiolaria), together forming a hitherto largely unrecognized major protozoan assemblage (core Rhizaria) in the eukaryote tree. Both 18S rRNA sequence trees and a rare deletion show that nonciliate haplosporidian and paramyxid parasites of shellfish (together comprising the Ascetosporea) are not two separate phyla, as often thought, but part of the Cercozoa, and may be related to the plant-parasitic plasmodiophorids and phagomyxids, which were originally the only parasites included in the Cercozoa. We discuss rRNA trees in relation to other evidence concerning the basal diversification and root of the eukaryotic tree and argue that bikonts and opisthokonts, at least, are holophyletic. Amoebozoa and bikonts may be sisters—jointly called anterokonts, as they ancestrally had an anterior cilium, not a posterior one like opisthokonts; this contrasting ciliary orientation may reflect a primary divergence in feeding mode of the first eukaryotes. Anterokonts also differ from opisthokonts in sterol biosynthesis (cycloartenol versus lanosterol pathway), major exoskeletal polymers (cellulose versus chitin), and mitochondrial cristae (ancestrally tubular not flat), possibly also primary divergences.     

Ovalopodium desertum n. sp. and the phylogenetic relationships of Cochliopodiidae (Amoebozoa)

An amoeba isolated from a weakly saline semi-desert pond in Kazakhstan (Central Asia) resembles a small Cochliopodium in the light microscope, but has a dorsal fibrous cell coat without scales. Thus it can be identified morphologically as a new species of Ovalopodium Sawyer, 1980, and it is herein named O. desertum. Phylogenetic analysis of the SSU rRNA gene sequences of the new species and four Cochliopodium spp. sequenced additionally shows that Ovalopodium desertum is a sister clade to a robustly monophyletic Cochliopodium. The close relationship between Ovalopodium and Cochliopodium is also confirmed by the analysis of SSU rRNA secondary structure showing the specific helices in the region V5 in all species of both genera. Analysis of actin gene sequences fails to resolve the position of Ovalopodium but demonstrates that Parvamoeba Rogerson, 1993 is probably related to Cochliopodium. The position of Cochliopodiidae within Amoebozoa remains unresolved, despite our efforts to resolve it using broader taxonomic sampling of Amoebozoa, testing alternative tree topologies and removing the fast-evolving sites. Among sequenced genera, Parvamoeba and Endostelium Olive et al., 1984 are probable relatives to Cochliopodiidae. Molecular trees weakly support an inclusion of the family in Flabellinia (Discosea), but more phylogenomic data are necessary to test this hypothesis.     

Evolutionary relationships of apusomonads inferred from taxon-rich analyses of 6 nuclear encoded genes

The phylogenetic relationships of the biflagellate protist group Apusomonadidae have been unclear despite the availability of some molecular data. We analyzed sequences from 6 nuclear encoded genes-small-subunit rRNA, large-subunit rRNA, alpha-tubulin, beta-tubulin, actin, and heat shock protein 90-to infer the phylogenetic position of Apusomonas proboscidea Aléxéieff 1924. To increase the taxon richness of the study, we also obtained new sequences from representatives of several other major eukaryotic groups: Chrysochromulina sp. National Institute for Environmental Studies 1333 (Haptophyta), Cyanophora paradoxa (Glaucophyta), Goniomonas truncata (Cryptophyceae), Leucocryptos marina (Kathablepharidae), Mesostigma viride (Streptophyta, Viridiplantae), Peridinium limbatum (Alveolata), Pterosperma cristatum (Prasinophytae, Viridiplantae), Synura sphagnicola (Stramenopiles), and Thaumatomonas sp. (Rhizaria). In most individual gene phylogenies, Apusomonas branched close to either of the 2 related taxa-Opisthokonta (including animals, fungi, and choanoflagellates) or Amoebozoa. Combined analyses of all 4 protein-coding genes or all 6 studied genes strongly supported the hypothesis that Apusomonadidae is closely related to Opisthokonta (or to all other eukaryotic groups except Opisthokonta, depending on the position of the eukaryotic root). Alternative hypotheses were rejected in approximately unbiased tests at the 5% level. However, the strong phylogenetic signal supporting a specific affiliation between Apusomonadidae and Opisthokonta largely originated from the alpha-tubulin data. If alpha-tubulin is not considered, topologies in which Apusomonadidae is sister to Opisthokonta or is sister to Amoebozoa were more or less equally supported. One current model for deep eukaryotic evolution holds that eukaryotes are divided into primary "unikont" and "bikont" clades and are descended from a "uniflagellate" common ancestor. Together with other information, our data suggest instead that unikonts (=Opisthokonta and Amoebozoa) are not strictly monophyletic and are descended from biflagellate ancestors.     

Acrylamide Alters Cytoskeletal Protein Level in Rat Sciatic Nerves

Occupational exposure and experimental intoxication with acrylamide (ACR) produce neuropathy characterized by nerve degeneration. To investigate the mechanism of ACR-induced neuropathy, male adult Wistar rats were given ACR (20, 40 mg/kg i.p. 3 days/week) for 8 weeks. Sciatic nerves were Triton-extracted and centrifuged at a high speed (100,000 × g) to yield pellet and supernatant fractions. The contents of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results showed that the three neurofilament (NF) subunits (NF-L, NF-M, NF-H) in both the pellet and the supernatant fraction decreased significantly (P < 0.01) in the high-dosing group, except for NF-M in the pellet. α-tubulin, β-tubulin, and β-actin increased significantly in the supernatant (P < 0.01), whereas both α-tubulin and β-tubulin decreased significantly in the pellet (P < 0.01). However, β-actin was not altered significantly in the sciatic nerves pellet. These findings suggest that ACR altered the cytoskeletal protein level in sciatic nerve, which may be one of the molecular mechanisms of ACR-induced peripheral neuropathy.     

Dynamics of cytoskeletal proteins in developing pine ectomycorrhiza

 Mycorrhizal short roots of Pinus contorta Dougl. ex Loud colonized by Suillus variegatus (Sow. ex Fr.) O. Kuntze or Paxillus involutus (Batsch) Fr. were collected 1–>60 days after fungal contact. The proteins of the inoculated roots were extracted, electrophoretically separated, blotted and immunostained for α-tubulin and actin. The development of the mycorrhiza was also followed microscopically. The signal of plant α-tubulin was stronger than the signal of fungal α-tubulin during the first 5 days in S. variegatus mycorrhiza and was then exceeded by fungal α-tubulin. This correlated well with the increase of fungal mycelium in the mycorrhiza. A transient drop in both plant and fungal α-tubulin signals was observed 20 days after fungal contact, suggesting a change in the metabolism of the mycorrhiza. The signals for plant and fungal actins in the mycorrhiza increased steadily during early infection and then remained at a high level as the mycorrhiza matured. Similar trends were observed in P. contorta–P. involutus mycorrhiza. The data from P. contorta–S.variegatus mycorrhizas suggests that α-tubulin is a growth-related protein, subject to changes, while the amount of actin reflects the general metabolic activity of the mycorrhiza. In both mycorrhizal systems clear α-tubulin and actin signals were detected 60 days after colonization, which indicates that the mycorrhizas were metabolically active in spite of their withered appearance. Accepted: 6 May 1996     

Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

Background  

Capping protein (CP), a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform.     

The evolutionary relationships among known life forms

Summary Sequences of small subunit (SSU) and large subunit (LSU) ribosomal RNA genes from archaebacteria, eubacteria, and the nucleus, chloroplasts, and mitochondria of eukaryotes have been compared in order to identify the most conservative positions. Aligned sets of these positions for both SSU and LSU rRNA have been used to generate tree diagrams relating the source organisms/organelles. Branching patterns were evaluated using the statistical bootstrapping technique. The resulting SSU and LSU trees are remarkably congruent and show a high degree of similarity with those based on alternative data sets and/or generated by different techniques. In addition to providing insights into the evolution of prokaryotic and eukaryotic (nuclear) lineages, the analysis reported here provides, for the first time, an extensive phylogeny of the mitochondrial lineage.     

Functional Significance May Underlie the Taxonomic Utility of Single Amino Acid Substitutions in Conserved Proteins

We hypothesized that some amino acid substitutions in conserved proteins that are strongly fixed by critical functional roles would show lineage-specific distributions. As an example of an archetypal conserved eukaryotic protein we considered the active site of β-tubulin. Our analysis identified one amino acid substitution—β-tubulin F224—which was highly lineage specific. Investigation of β-tubulin for other phylogenetically restricted amino acids identified several with apparent specificity for well-defined phylogenetic groups. Intriguingly, none showed specificity for “supergroups” other than the unikonts. To understand why, we analysed the β-tubulin Neighbor-Net and demonstrated a fundamental division between core β-tubulins (plant-like) and divergent β-tubulins (animal and fungal). F224 was almost completely restricted to the core β-tubulins, while divergent β-tubulins possessed Y224. Thus, our specific example offers insight into the restrictions associated with the co-evolution of β-tubulin during the radiation of eukaryotes, underlining a fundamental dichotomy between F-type, core β-tubulins and Y-type, divergent β-tubulins. More broadly our study provides proof of principle for the taxonomic utility of critical amino acids in the active sites of conserved proteins.     

Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing α-sarcomeric actin, desmin, β-tubulin, spectrin/α-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of α-sarcomeric actin and spectrin/α-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/α-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas β-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/α-fodrin fibres as well as actin and desmin filaments. Accepted: 28 July 1999     

Role of Cytoskeleton Proteins in the Morphological Changes During Apoptotic Cell Death of Cerebellar Granule Neurons

Cytoskeleton proteins are substrates for proteases and further apoptotic death. We evaluated the participation of cytoskeleton in morphological changes during cell death induced by two apoptotic conditions, potassium deprivation (K5) and staurosporine, in cerebellar granule neurons (CGC). We found that K5 induced somatic damage, but neurites were relatively preserved, which corresponded to the reorganization of actin and α-tubulin in neurites. Staurosporine (STS) induced an early alteration of neurites with reorganization of cytoskeleton proteins in somas. Caspase inhibitor ZVAD totally inhibited STS-induced α-tubulin reorganization and partially blocked STS-induced actin reorganization. α-tubulin and actin reorganization induced by K5 was affected by ZVAD. Calpain inhibitor (IC1) did not affect α-tubulin or actin reorganization induced by STS, K5 or ionomycin. Neither ZVAD, nor IC1 changed α-tubulin or actin levels upon K5 treatment. STS increased α-tubulin and actin levels, but neither ZVAD nor IC1 changed α-tubulin levels upon STS treatment. In contrast, ZVAD reduced the STS-induced increase of actin. These results suggest that CGC cytoskeleton proteins undergo a differential expression and reorganization depending on the apoptotic condition.     

Evidence for myxobacterial origin of eukaryotic defensins

Antimicrobial defensins with the cysteine-stabilized α-helical and β-sheet (CSαβ) motif are a large family of ancient, evolutionarily related innate immunity effectors of multicellular organisms. Although the widespread distribution in plants, fungi, and invertebrates suggests their uniqueness to Eukarya, it is unknown whether these eukaryotic defensins originated before or posterior to the emergence of eukaryotes. In this study, we provide evidence in support of the existence of defensin-like peptides (DLPs) in myxobacteria based on structural bioinformatics analysis, which recognized two bacterial peptides with a conserved cysteine-stabilized α-helical motif, a nested structural unit of the CSαβ motif. Similarity in sequence and structure to fungal DLPs together with restricted distribution to the myxobacteria as well as central role of the myxobacteria in the origin of eukaryotes suggest that the bacterial DLPs represent the ancestor of the eukaryotic defensins and could mediate immune defense of early eukaryotes after gene transfer to the proto-eukaryotic genome. Our work thus offers a basis for further investigation of prokaryotic origin of eukaryotic immune effector molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

STRUCTURAL AND SEQUENCE SIGNATURES OF NUCLEAR SSU RRNA DEFINE TAXONOMIC LEVELS WITHIN THE RHODOPHYTA

Over 400 nuclear SSU rRNA sequences representing all orders of the Rhodophyta were aligned and analyzed using comparative sequence analysis. Numerous nucleotide positions and structural elements were found that delineated various taxonomic groups. The 1245 region (E. coli numbering) contained a loop that differed in size between two conserved helices and clearly separated the Florideophyceae [3 nt (>95% of 268 sequences)], Bangiales [13 to 14 nt (100% of 116 sequences)] and remaining Bangiophyceae including the Cryptophyta nucleomorphs [four to eight nt (100% of 32 sequences)]. In addition, members of the Thoreaceae were found to have additional helices in the 650 and 1139 region of which a corresponding structure was not present in any other red algal SSU rRNA gene sequence. Base‐pair and nucleotide signatures differentiated the Bangiales, Florideophyceae, Bangiophyceae (not including Bangiales) and Hildenbrandiales at three levels of comparison: within the Rhodophyta (>400 sequences), the Eukaryota (not including Rhodophyta;> 1300 sequences) and three kingdom (Archaea, Bacteria, 2 organelles, Eukaryota;> 7000 sequences). For example, all members of the Hildenbrandiales have a change in the base‐pair 512:539 that is a region of functional importance. Sequences from the Eukaryota, Archaea, Bacteria and two organelles have a C:G or a U:A in this position whereas the Hildenbrandiales have a C:A pair. This analysis raises the possibility of utilizing structural features of nuclear SSU rRNA and sequence signatures to support and delineate phylogenetic clades within the Rhodophyta.