The Chaperonin Genes of Jakobid and Jakobid-Like Flagellates: Implications for Eukaryotic Evolution

John M. Archibald1, Charles J. O'Kelly and W. Ford Doolittle

*Department of Biochemistry and Molecular Biology, Dalhousie University;
{dagger}Bigelow Laboratory for Ocean Sciences


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs—similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The jakobid flagellates are free-living, mitochondriate, heterotrophic protists (O'Kelly 1993, 1997Citation ). They include the families Jakobidae, genus Jakoba Patterson, 1990Citation (Patterson 1990Citation ) and Histionidae, genera Histiona Voigt, 1901, and Reclinomonas Flavin and Nerad, 1993Citation (Flavin and Nerad 1993Citation ). The jakobids have figured prominently in hypotheses about the origin and early evolution of eukaryotes, originally because of the presence of ultrastructural features shared with certain amitochondriate lineages (O'Kelly 1993Citation ). Small subunit ribosomal RNA (SSUrRNA) and protein phylogenies often place the amitochondriates at or near the base of the eukaryotic tree (e.g., Sogin et al. 1989Citation ; Leipe et al. 1993Citation ; Hashimoto et al. 1994Citation ; Stiller et al. 1998Citation ); O'Kelly (1993)Citation hypothesized that if amitochondriate protists were the earliest diverging eukaryotic lineages, then the earliest diverging mitochondriate eukaryotes should share ultrastructural features with these groups.

The sequencing of jakobid mitochondrial genomes has provided startling insight into mitochondrial evolution and has further suggested a pivotal role for the jakobids in our understanding of the evolution of eukaryotes. These organisms possess the most bacterial-like mitochondrial genomes characterized thus far (Lang et al. 1997Citation ; Gray et al. 1998Citation ; Gray, Burger, and Lang 1999Citation ). Most striking is the mitochondrial DNA (mtDNA) of Reclinomonas americana, which encodes 97 genes, more than are present in any other mitochondrial genome. Several of these have never before been found encoded in mtDNA, including a gene for a bacterial translation factor (tufA), a putative cytochrome oxidase assembly protein (cox11), a secretion pathway protein (secY), and genes for four subunits of a bacterial-type RNA polymerase (rpoA-D; single subunit phage-type RNA polymerases are thought to function in all other known mitochondria) (Lang et al. 1997Citation ; Gray et al. 1998Citation ). Operon-like ribosomal protein gene clusters similar to those found in bacteria are also present.

More recently, the jakobids have been considered members of a much larger assemblage of protists, the excavate taxa. The excavates are a diverse group of amitochondriate, mitochondriate, and hydrogenosomal lineages that share as their uniting feature the presence of a ventral feeding groove (Patterson, Simpson, and Weerakoon 1999Citation ; Simpson and Patterson 1999Citation ). In addition to the jakobids (Jakoba, Reclinomonas, and Histiona) and the jakobid-like nanoflagellate Malawimonas (O'Kelly and Nerad 1999Citation ), the excavates include the heteroloboseans, diplomonads, retortamonads, Trimastix, and Carpediemonas (Simpson and Patterson 1999Citation ). Whereas ultrastructural data suggest that these organisms share a common excavate (i.e., feeding groove bearing) ancestor, there is no consensus view on the relationships amongst the various excavate taxa or their relationship to other mitochondriate, amitochondriate, and hydorogenosome-containing groups. Indeed, current views on the origin and evolution of eukaryotes are in a state of flux. Many of the putatively deep-branching and primitively amitochondriate protist lineages (including some of the excavate taxa) are now thought to be derived from mitochondrion-bearing ancestors (see Roger 1999Citation for recent review). Further, the ability of current phylogenetic methods to accurately reconstruct the deepest branches of phylogenetic trees has come into question (Hirt et al. 1999Citation ; Stiller and Hall 1999Citation ; Philippe and Germot 2000Citation , and references therein). Philippe and Adoutte (1998)Citation suggested that a big bang occurred at the base of eukaryotes and that the major cladogenetic events in eukaryotic evolution occurred in quick succession. Thus, there is at present no clear picture as to which protist groups—if any—actually represent early diverging lineages.

We are studying the evolution of chaperonins in diverse amitochondriate and mitochondriate protists. Group II chaperonins, a class of molecular chaperone found in Archaea and the eukaryotic cytosol, are double-ring protein complexes that mediate the proper folding of nascent or denatured proteins (for review see Willison and Horwich 1996Citation ). The eukaryotic cytosolic chaperonin complex (CCT or TriC) is best known for its role in mediating the proper folding of the cytoskeletal proteins actin and tubulin (Willison and Kubota 1994Citation ; Kubota, Hynes, and Willison 1995Citation ). Recent experiments suggest that a large number of newly translated proteins may also interact with CCT (Melki et al. 1997Citation ; Thulasiraman, Yang, and Frydman 1999Citation ). Unlike bacterial and organellar chaperonins, which are usually homo-oligomeric, the eukaryotic CCT complex contains eight different homologous subunits, alpha, beta, gamma, delta, epsilon, zeta, eta, and theta (Willison and Horwich 1996Citation ). The duplications producing the different CCT genes are known to have occurred prior to the divergence of the parabasalids and diplomonads from other eukaryotes (Archibald, Logsdon, and Doolittle 2000Citation ). Individual CCT subunits are over 500 amino acids in length and are highly conserved, both desirable properties for a phylogenetic marker. The presence of eight distinct CCT paralogs makes it possible to perform multiple reciprocal rootings of the phylogenetic tree of eukaryotes. Unlike SSUrRNA, EF-1{alpha}, actin, and tubulin, the CCTs are relatively poorly sampled. Focusing on the alpha subunit of CCT, we therefore sought to broaden the diversity of taxonomic representation and add to the Trichomonas vaginalis and Giardia lamblia CCTalpha's sequenced in a recent study (Archibald, Logsdon, and Doolittle 2000Citation ).


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Genomic DNAs
The R. americana (ATCC number 50394 and 50283) and M. jakobiformis (ATCC number 50310) genomic DNAs (gDNAs) used in this study were kindly provided by Dr. B. F. Lang (Université de Montréal, Canada). Dr. S. L. Hajduk and M. Oli (University of Alabama) provided gDNA from Trypanosoma brucei (strain MiTat 1.2 427/221), Dr. A. J. Roger (Dalhousie University, Canada) provided Acrasis rosea gDNA, and Naegleria gruberi gDNA was a gift from Dr. R. J. Redfield (University of British Columbia, Canada), Dr. J. M. Logsdon Jr. (Emory University), and J. Dacks (Dalhousie University, Canada).

Cloning and Sequencing of CCT Genes
Degenerate PCR primers were used to amplify CCT genes from gDNA. A combination of universal chaperonin primers (Archibald, Logsdon, and Doolittle 2000Citation ) and CCTalpha-specific primers were used to amplify the CCTalpha gene (forward primers: CCT-2-for [5'-AACGACGGTGCNACNATHYT-3'], CCT-9-for [5'-CCAGTCGGTCTNGAYAARATG-3']; reverse primers: CCT-4-rev [5'-CTCTACAGCNCCNSCNCC-3'], CCT-10-rev [5'-TGATCAGRTCRTCDATNC-3'], CCT-11-rev [5'-AGGTCGTCGATGCKNARDAT-3'], TF-9-rev [GCAGCTATCARRTCRTCDAT-3']). With most primer combinations, 90%–95% of the gene was amplified. The CCTdelta gene was amplified with CCT-9-for (above) and CCT-7-rev [5'-ACGATGCACATNGHRTCRTG-3'].

PCR reactions were carried out on an MJ Research Inc. PTC-100 thermal cycler using GIBCO-BRL Taq polymerase, buffer, and dNTP. The PCR reactions performed with R. americana and M. jakobiformis DNAs contained 5% acetamide (final concentration). Following an initial denaturation of 3 min at 92°C, reactions were performed with 40–45 cycles of 92°C for 15 s, 50–54°C for 30 s, and 72°C for 3 min. PCR products of the expected size were cloned directly from low-melt agarose with the TOPO-TA cloning kit (Invitrogen). Clones were screened for the presence of inserts by EcoRI restriction of isolated plasmid DNAs or by PCR-screening directly from E. coli cells with the M13 universal forward and reverse primers. Sequencing was performed manually (T7 sequencing kit, Pharmacia) and with LiCor and ABI automated sequencers.

CCT sequences from Plasmodium falciparum were identified by BLAST (Altschul et al. 1990Citation ) at the PlasmoDB website (http://www.plasmodb.org/). CCTalpha was obtained from chromosome 11 sequence, whereas CCTdelta was found in genomic sequence from chromosome 13. Preliminary sequence data for P. falciparum chromosomes 10 and 11 was obtained from The Institute for Genomic Research website (www.tigr.org). Sequencing of chromosomes 10 and 11 was part of the International Malaria Genome Sequencing Project and was supported by award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Sequence data for P. falciparum chromosome 13 was obtained from The Sanger Centre website at http://www.sanger.ac.uk/Projects/P_falciparum/. Sequencing of P. falciparum chromosome 13 was accomplished as part of the Malaria Genome Project with support by The Wellcome Trust.

For T. brucei, a small fragment of the CCTalpha gene was found by searching genomic data from unfinished microbial genomes. Preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org. Based on this sequence an exact-match reverse primer (Tbru.CCTa.R-1 [5'-GTTAAGGCGAACACAGTCAT-3']) was designed and used in combination with degenerate forward primers (CCT-2-for, CCT-9-for; above) to amplify most of the CCTalpha coding sequence from T. brucei gDNA. From the sequence of five independent clones, two different CCTalpha clones were apparent, one of which matched the original fragment present in the T. brucei genome data. The ambiguities between the two clone types were almost all synonymous substitutions, suggesting the presence of multiple copies of the CCTalpha gene in T. brucei (see Results). The sequences presented in this study have been deposited in GenBank under the following accession numbers: AF322043AF322050.

Southern Hybridization
To confirm the source and copy number of the CCT genes, PCR products obtained from R. americana (ATCC number 50394) and M. jakobiformis (ATCC number 50310) were isolated (BIORAD, Prep-a-gene), labeled with {alpha}32P (Prime-It II random primer labeling kit, Stratagene), and hybridized to restricted gDNA.

Sequence Characterization and Phylogeny
A combination of BLAST and DNA Strider (Douglas 1995Citation ) was used to determine the positions of introns in the new CCT genes and in CCTs retrieved from the public databases. Inferred amino acid sequences from the Reclinomonas, Malawimonas, heterolobosean, Trypanosoma and Monocercomonas CCTs were added manually, based on globally conserved regions, to an alignment constructed previously (Archibald, Logsdon, and Doolittle 2000Citation ). From this master data set, smaller alignments containing subsets of the data were constructed. Each alignment was assessed individually and regions of ambiguity were removed to ensure that only confidently aligned amino acid positions were used for phylogenetic reconstruction. Gaps for missing data (e.g., the extreme N- and C-termini missing from PCR-generated sequences) were also removed. The complete CCTalpha-only alignment contained 26 taxa and 424 unambiguously aligned amino acid positions. The data sets for rooted analyses contained 23 CCTalpha sequences (three highly similar mammalian CCTs were removed) and between five and eight outgroup sequences chosen for maximal taxonomic diversity. These data sets contained the following number of sites: CCTalpha-beta—381 sites, CCTalpha-gamma—360 sites, CCTalpha-delta—378 sites, CCTalpha-eta—369 sites, CCTalpha-epsilon—351 sites, CCTalpha-theta—334 sites, and CCTalpha-zeta—357 sites. All alignments are available from J.M.A. upon request (jarch@interchange.ubc.ca).

Phylogenetic trees were inferred from amino acid sequences using maximum likelihood (ML) and ML-distance methods of tree reconstruction. ML analyses were performed with the following programs: proML in PHYLIP 3.6 (http://evolution.genetics.washington.edu/phylip.html), using the Dayhoff amino acid substitution matrix, the global rearrangements option, a randomized sequence input order (10 jumbles), and an among-site rate variation (ASRV) model with eight rate categories plus an invariable rate category (the relative rates for each category were estimated in PUZZLE 4.02 [Strimmer and von Haeseler 1997Citation ]); protML (using the JTT-F amino acid substitution matrix) in MOLPHY (Adachi and Hasegawa 1996Citation ); quartet puzzling in PUZZLE 4.02, accounting for ASRV with an eight rate-category discrete approximation to the {Gamma} distribution plus an invariable rate category. ML-distance trees were inferred from {Gamma}-corrected distance matrices calculated in PUZZLE 4.02 using FITCH (with global rearrangements) in PHYLIP, version 3.57 (Felsenstein 1993Citation ). Support for proML trees was obtained by bootstrapping (100 replicates). For protML trees, RELL values (obtained from quick-add searches of the best 1,000 or 2,000 trees in protML options -q -n 1,000, 2,000; Adachi and Hasegawa 1996Citation ) were used as measures of statistical support. Support values for ML-distance trees were obtained by bootstrapping (500 replicates) with PUZZLEBOOT 1.02 (A. Roger and M. Holder; http://members.tripod.de/korbi/puzzle/). PUZZLE was used to statistically assess the significance of different tree topologies using the Kishino-Hasegawa test (Kishino and Hasegawa 1989Citation ).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Jakobid and Malawimonad CCT Genes Possess Numerous Spliceosomal Introns
We used a degenerate PCR approach to amplify the CCTalpha gene from R. americana, M. jakobiformis, two heteroloboseans (A. rosea and N. gruberi), the euglenozoan T. brucei, and the parabasalid Monocercomonas sp. We also amplified a somewhat smaller fragment of the CCTdelta gene from M. jakobiformis. Repeated attempts to amplify CCTalpha from Jakoba libera (a jakobid) were unsuccessful, despite the use of all possible primer combinations under a wide range of conditions. PCR reactions using additional degenerate primers designed with a strong bias toward G + C at the third codon position (see below) also failed.

The jakobid-malawimonad CCT genes presented here are among the first nuclear protein-coding genes to be characterized from these protists. The most striking feature of the R. americana and M. jakobiformis CCTs is the presence of multiple spliceosomal introns. The CCTalpha gene from R. americana possessed five introns (ranging from 67 to 145 nt in length, with some size heterogeneity between homologous introns in the two strains), whereas the M. jakobiformis CCTalpha contained seven introns (58–127 nt long). The M. jakobiformis CCTdelta gene possessed seven introns between 61 and 73 nt in length, despite being a shorter fragment of coding sequence than that obtained for CCTalpha (~1 kb of ORF). When the putative introns were removed, the inferred protein sequences were readily alignable with orthologs from a wide range of other eukaryotes, with no size heterogeneity at the intron-exon boundaries. All the introns possessed standard 5'-GT...AG-3' intron boundaries, with the exception of a single intron in one of the two CCTdelta clones from M. jakobiformis, which possessed a CT at the 5' intron-exon boundary. It is unclear whether this represents a PCR-generated artifact or a legitimate noncanonical 5' intron boundary. A summary of the sizes and positions of the introns found in the R. americana and M. jakobiformis CCT genes is shown in figure 1 . With the exception of a single intron in the A. rosea CCTalpha, none of the nonjakobid-nonmalawimonad CCTalpha genes sequenced in this study contained introns.



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Fig. 1.—Spliceosomal introns in the Reclinomonas and Malawimonas CCT genes. The schematic shows the positions and sizes of the introns found in the R. americana and M. jakobiformis CCTalpha genes and the M. jakobiformis CCTdelta gene

 
The sequencing of independent clones suggested the presence of multiple copies of the CCTalpha gene in R. americana (50283 and 50394), N. gruberi, T. brucei, and Monocercomonas sp., as well as the CCTdelta gene in M. jakobiformis. Between the different clone types, most of the observed substitutions were synonymous; however, slightly different amino acid sequences were inferred from different clones of the Monocercomonas sp. CCTalpha and M. jakobiformis CCTdelta genes. Some size heterogeneity between homologous introns in the different clones was also observed. The presence of multiple copies of CCT genes was subsequently confirmed for CCTalpha in R. americana 50394 and for the M. jakobiformis CCTdelta by the southern hybridization of PCR products to restricted gDNAs (data not shown). Overall, a strong G + C bias was present in the R. americana and M. jakobiformis CCT genes, particularly at the third codon position. Edgcomb et al. (2001)Citation observed a similar bias in jakobid tubulins.

CCTalpha Phylogeny
In an attempt to determine the relationships between the jakobid and malawimonad flagellates and their relationship to other eukaryotes, we performed phylogenetic analyses on the CCTalpha data set. We also performed multiple rooted analyses using the seven other CCT paralogs to root the CCTalpha tree. Figure 2 shows a ML-distance phylogeny inferred from CCTalpha protein sequences. Interestingly, the jakobid R. americana and the jakobid-like M. jakobiformis showed no affinity for one another in the CCTalpha tree. Whereas the phylogenetic position of M. jakobiformis with respect to the other protists was quite unstable, R. americana consistently (but weakly) branched with the two heteroloboseans (as in fig. 2 ) and only occasionally with T. brucei (in proML analyses; data not shown). In many ways, the CCTalpha topology is similar to those obtained with more widely used phylogenetic markers, such as SSUrRNA, actin, EF-1{alpha}, and alpha- and beta-tubulin. For example, the sisterhood of animals and fungi, strongly supported by an ever-increasing wealth of molecular data (see Baldauf 1999Citation for recent review) is moderately well supported with all phylogenetic methods. Also, the single mycetozoan representative in the CCTalpha data set, Dictyostelium discoideum, branches weakly but consistently at the base of animals and fungi, in line with suggestions that the Mycetozoa are an outgroup to the animal-fungal clade (Baldauf 1999Citation ). The alveolates, represented in our data set by the ciliate Tetrahymena pyriformis and the apicomplexan parasite P. falciparum, receive reasonable support as a monophyletic grouping, and as expected, the two heterolobosean sequences (A. rosea and Nagleria gruberi) branch together with strong support and with all phylogenetic methods. The parabasalid CCTalphas (Monocercomonas sp., sequenced here and T. vaginalis, sequenced previously; Archibald, Logsdon, and Doolittle 2000Citation ) are characterized by extremely long branches, similar to phylogenies constructed with other molecules.



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Fig. 2.—Phylogeny of CCT alpha protein sequences. The tree shown is a distance (Fitch-Margoliash) tree inferred from a ML distance matrix calculated with a rate heterogeneity model (JTT-F + {Gamma} + inv, proportion of invariable sites = 0.08, {Gamma} distribution shape parameter alpha = 1.23). The tree was constructed using a data set containing 26 taxa and 424 unambiguously aligned amino acid positions. Sequences determined in this study are highlighted. Support values for important nodes are ML RELL values and ML-distance bootstrap values, respectively. The scale bar indicates the inferred number of substitutions per site

 
The CCTalpha topology differs from other molecular phylogenies of eukaryotes in several interesting ways. The placement of the alveolates near the parabasalids is not seen with most phylogenetic markers but is reminiscent of phylogenies of actin, where the ciliate sequences are paraphyletic and branch near the base of the tree (e.g., Bricheux and Brugerolle 1997Citation ). Also, the amitochondriate diplomonad G. lamblia branches with the mitochondriate euglenozoan T. brucei. Together, these taxa form a weakly supported but consistently observed clade with the heteroloboseans (N. gruberi and A. rosea) and the two R. americana strains. Despite the fact that the Giardia CCTalpha is a fairly long branch, this relationship was still observed in the majority of rooted analyses (see below).

Given the general lack of resolution among the protists in the CCTalpha phylogeny, we examined the significance of alternate topologies using the Kishino-Hasegawa test (Kishino and Hasegawa 1989Citation ), in particular, the relative positions of the R. americana and M. jakobiformis sequences. Interestingly, trees in which R. americana and M. jakobiformis were specific sister groups were found to be worse than the topology shown in figure 2 at a 5% level of significance, as were trees in which M. jakobiformis was placed as an outgroup to the R. americana-Heterolobosea-Giardia-Trypanosoma clade. We also tested the effect of removing various long-branch taxa on the support for the topology obtained with the full CCTalpha data set. When the parabasalids were removed, the overall topology was the same, except for the placement of M. jakobiformis, which moved to a position adjacent to the alveolates (data not shown). Interestingly, the support for the R. americana-Heterolobosea grouping increased with most phylogenetic methods, as did the support for Giardia branching with T. brucei (data not shown).

When CCTalpha was rooted with each of the seven other CCT paralogs, different topologies were often obtained from different data sets and from the same data set analyzed with different phylogenetic methods. However, the parabasalids were usually the deepest branch in the CCTalpha tree, and the alveolates were often the next deepest branch (sometimes as a paraphyletic group). Figure 3A shows the CCTalpha-theta tree, which represents the most commonly obtained topology with the various methods and data sets. Significantly, G. lamblia was not attached to the long branch of the outgroup in most analyses but grouped with T. brucei. Overall, the internal topology was very similar to that obtained in the unrooted analyses (fig. 2 ). Figure 3B shows the CCTalpha-beta tree, where the G. lamblia sequence branches near the base of CCTalpha, and R. americana branches with T. brucei and not with the heteroloboseans.



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Fig. 3.—Rooted analyses of CCTalpha. A, Majority. The CCTalpha-theta tree showing the CCTalpha topology most often observed when rooted with another CCTalpha paralog. The tree shown is the ML tree obtained from a heuristic search of 1,000 trees in protML (lnL = -11253.17), with branch lengths reestimated in PUZZLE under an ASRV model (the proportion of invariable sites was 0.03 and the {Gamma} distribution shape parameter alpha = 1.52). The tree was constructed from an alignment containing 334 unambiguously aligned amino acid positions. B, Minority. The CCTalpha-beta tree, an example of an alternate CCTalpha topology. Tree shown is the ML-distance tree inferred with the Fitch-Margoliash algorithm from an ML distance matrix calculated with an ASRV model (JTT-F + {Gamma} + inv, proportion of invariable sites = 0.08, {Gamma} distribution shape parameter alpha = 1.49). The alignment contained 381 sites. Support values are provided if greater than 40% (ML RELL values and ML-distance bootstrap values, respectively). Dashed lines between the trees in A and B highlight differences in the deepest branches of the two CCTalpha topologies. The scale bars indicate the inferred number of amino acid substitutions per site

 
Intron Phylogeny
The density of spliceosomal introns in the Reclinomonas and Malawimonas CCT genes is extraordinarily high—on par with that observed in vertebrate genes (Logsdon 1998Citation ). To compare the diversity of intron positions in the jakobid and malawimonad CCTs to those in animals, fungi, plants, and other protists, we surveyed all available CCTalpha and CCTdelta genomic sequences. The results are presented in figure 4A and B . From the 17 CCTalpha sequences for which genomic sequence data was available, 35 distinct intron positions were observed. For CCTdelta, 14 sequences were available for comparison, and 21 intron positions were found. Interestingly, three out of the five introns present in the R. americana CCTalpha gene were shared with M. jakobiformis, despite the fact that the two groups showed no affinity for one another in phylogenies of CCTalpha (above), tubulin (Edgcomb et al. 2001Citation ), or concatenated mitochondrial proteins (Burger et al. 1999, 2000Citation ). Of these three, one was shared with A. thaliana and two of three animals (position 21) and another with A. thaliana and the heterolobosean A. rosea (position 10). One intron (position 2) was conserved between protists (P. falciparum and T. pyriformis), plants, one of two fungi, and animals (fig. 4A ). For CCTdelta, two introns were shared between at least one protist and two of four animals (positions 5 and 6) and a third between M. jakobiformis, one of three fungi (Neurospora crassa), and animals (position 13) (fig. 4B ). Despite the general high abundance of introns in plant protein-coding genes (Logsdon 1998Citation )—16 introns were found in the Arabidopsis CCTalpha gene—neither of the plant CCTdeltas (Arabidopsis or Oryza) contained any introns. This likely represents a case of recent retrointegration of a cDNA, although in the absence of additional data, it is difficult to say how recent. A large number of unique intron positions are also present in both data sets. Twenty-four of 35 CCTalpha introns and 9 of 21 CCTdelta introns were present in a single species (considering the two R. americana strains as a single group), although for CCTalpha, 12 of the 24 introns were unique to A. thaliana. Two of the seven CCTalpha and four of the seven CCTdelta introns in M. jakobiformis were not present in any other taxa.



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Fig. 4.—CCTalpha and CCTdelta intron phylogenies. A, Phylogenetic distribution of 35 known CCTalpha introns. The tree shown is a consensus topology of rooted and unrooted phylogenies of CCTalpha protein sequences (figs. 2, 3A, and 3B ) showing only strongly supported relationships. A plus (+) sign indicates the presence of an intron, a minus indicates intron absence, and a question mark indicates missing data. Intron positions are numbered and potentially conserved intron positions are highlighted. B, Phylogenetic distribution of 21 known CCTdelta introns. Topology shown is a consensus of CCTdelta phylogenetic analyses (data not shown). For both data sets, only introns in exactly the same codon phase were considered to be in the same position. Sequences determined in this study are in bold. AN, animals; FU, fungi; PL, plants; PR, protists

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Until recently, attempts to determine the phylogenetic affinities of the jakobid and jakobid-like flagellates to one another and to other protists have relied solely on their ultrastructural features. The analyses presented here are among the first to characterize nuclear protein-coding genes from these organisms and are the first to assess the utility of the cytosolic chaperonin CCTalpha as a marker for eukaryotic phylogeny. Whereas the failure to obtain a CCTalpha sequence from J. libera (see Results) precludes any direct comparisons between J. libera, R. americana, and M. jakobiformis, the latter two show no detectable phylogenetic affinity for one another in our analyses. This result is consistent with a recent analysis of jakobid alpha- and beta-tubulins (Edgcomb et al. 2001Citation ) and with phylogenies constructed from concatenated mitochondrial proteins (Burger et al. 1999, 2000Citation ). In the CCTalpha phylogenies M. jakobiformis showed no affinity for any particular protist group, whereas R. americana branched weakly but consistently with the heteroloboseans and the euglenozoan T. brucei (see below). A close relationship between R. americana and M. jakobiformis was also rejected using the Kishino-Hasegawa test (Kishino and Hasegawa 1989Citation ), although this result should be interpreted with caution. The test is known to be biased toward rejection when the best ML tree inferred from the data is included among the topologies tested (Goldman, Anderson, and Rodrigo 2000Citation ). A modified version of the test developed by Shimodaira and Hasegawa (1999)Citation aims to eliminate this problem, but at present there is debate over its proper implementation (Shimodaira and Hasegawa 1999Citation ; Goldman, Anderson, and Rodrigo 2000Citation ).

The weak but consistent affinity of R. americana for the heteroloboseans and euglenozoa in CCTalpha phylogenies (figs. 2, 3A, and 3B ) is interesting for two reasons. First, it is consistent with beta-tubulin and combined alpha-beta–tubulin trees (Edgcomb et al. 2001Citation ). Second, it is consistent with mitochondrial gene-content data. The mtDNA of the heterolobosean N. gruberi contains (in addition to many genes shared with plant and protist mtDNAs) two genes thus far only identified in jakobid mitochondrial genomes (atp3 and cox11; M. W. Gray, personal communication; http://megasun.bch.umontreal.ca/ca.ogmp/projects/ngrub/gen.html). Whereas the grouping of R. americana with the Heterolobosea is consistent with beta-tubulin and combined alpha-beta–tubulin phylogenies (Edgcomb et al. 2001Citation ) and mitochondrial gene-content data, it is at odds with mitochondrial cristal morphology. Like N. gruberi and the euglenozoan T. brucei, M. jakobiformis possesses discoidal cristae, yet it is R. americana, which possesses tubular mitochondrial cristae, that shows the most affinity for the Heterolobosea and T. brucei in the CCTalpha and tubulin trees. Whereas cristal morphology has often been taken to be an evolutionarily stable character, these data suggest that it may not always be reliable for tracking the deepest divisions in eukaryotic evolution. More generally, the significance of the retention of ancestral features in jakobid and malawimonad mtDNAs, in terms of their relationship to other eukaryotes, is as yet unclear. The M. jakobiformis mtDNA possesses three genes (rpl18, 19, 31) that have thus far not been observed in nonjakobid protist mtDNAs (http://megasun.bch.umontreal.ca/ogmp/projects/mjako/gen.html), yet all phylogenetic analyses performed thus far on both mitochondrial and nuclear genes suggest that M. jakobiformis and the jakobids do not form a monophyletic group.

On the whole, CCTalpha phylogenies show a great deal of congruence with more commonly used phylogenetic markers: animals and fungi appear to be each other's closest relatives, the mycetozoan Dictyostelium appears as an immediate outgroup to the animal-fungal clade, and the alveolates and Heterolobosea are monopyletic groups. Most interesting was the placement of the diplomonad G. lamblia. Almost without exception, rooted SSUrRNA and protein phylogenies place diplomonads at or near the base of the eukaryotic tree (Roger 1999Citation ). In most rooted analyses of CCTalpha, Giardia was not positioned at the base of the eukaryotic tree but was nested within mitochondrion-containing groups. This is consistent with the suggestion that diplomonads have lost their mitochondria secondarily (Roger et al. 1998Citation ; Roger 1999Citation ). The analyses presented here illustrate the problem with deep phylogeny, long-branch attraction (e.g., Germot and Philippe 1999Citation ; Stiller and Hall 1999Citation ; Philippe and Germot 2000Citation ). It is clear that the two parabasalids in the CCTalpha data set have the longest branches of all the taxa—they also emerge consistently at the base of the CCT tree in rooted analyses. For this reason, the deepest branches of the rooted CCTalpha phylogenies presented here should be viewed with caution.

The high density of introns in the jakobid and malawimonad CCT genes raises the question of their origin. Were they recently acquired, or do they represent the retention of old introns? Separate from the issue of the origin of introns themselves (i.e., introns-early vs. introns-late; Logsdon et al. 1995Citation ; de Souza et al. 1998Citation ; Logsdon 1998Citation ) is the question of the diversity and antiquity of spliceosomal introns within eukaryotes. For the most part, protist genes are intron sparse, and many of the lineages that have figured prominently in hypotheses of early events in eukaryotic evolution (e.g., diplomonads and parabasalids) seem to lack introns entirely (Logsdon 1998Citation ). Remarkably few intron positions are known to be conserved between animals, fungi, and plants (Fast, Logsdon, and Doolittle 1999Citation ; Palmer and Logsdon 1991Citation ) and fewer still between animals or fungi (or both), plants, and protists. Interestingly, the CCTalpha data set contains two introns of the latter sort (positions 2 and 21), and CCTdelta contains an intron shared between M. jakobiformis, one of three fungi, and animals (position 13). Unfortunately, the CCTalpha data set is somewhat biased against the presence of old introns because of the fact that the sole fungal representatives, Saccharomyces cerevisiae and Schizosaccharomyces pombe, are known to be relatively intron sparse compared to other fungi (the intron density in S. cerevisiae is only 0.1 per kb; Logsdon 1998Citation ). In the CCTdelta data set, three introns were found in the N. crassa CCTalpha (N. crassa has a higher intron density), and two of these were shared with animals. The CCTdelta data set is similarly biased. No introns were found in the S. cerevisiae or S. pombe CCTdelta genes, and despite the fact that the intron density in plant protein-coding genes is generally quite high (Logsdon 1998Citation ), neither of the plant CCTdeltas (Arabidopsis or Oryza) contained a single intron. With few protist introns available for comparison, it is difficult to make strong inferences about whether the potentially old jakobid-malawimonad introns are a result of the retention of ancestral intron positions or because of independent parallel insertions. More CCTalpha sequences from each of the major eukaryotic groups may resolve this issue.

It is certainly clear from both data sets that recent intron gain has occurred. The Arabidopsis thaliana CCTalpha gene alone contained 12 introns in positions not found in any other taxa, and the CCTalpha from the ciliate T. pyriformis contained another three (fig. 4A , see Results). For the jakobid-malawimonad CCTalpha's, one of the R. americana introns and two of the M. jakobiformis introns were located in unique positions, as were four of seven M. jakobiformis CCTdelta introns. Data from the jakobid tubulin genes also support this notion. As predicted from ultrastructure and mitochondrial genome content data, R. americana and J. libera branched strongly together in beta- and alpha-beta–tubulin phylogenies. Yet despite this apparent close relationship, their respective beta-tubulins each possessed a single intron located in different positions, neither of which is present in known beta-tubulin genes (Edgcomb et al. 2001Citation ).

As for intron loss, the data are more ambiguous. In general, this uncertainty stems from the lack of a robust phylogeny of the major protist groups upon which intron gain-loss scenarios can be evaluated. Nevertheless, a case for intron loss can be inferred for the Heterolobosea. Consistent with the low intron density observed thus far for heterolobosean protein-coding genes (Logsdon 1998Citation ), only one intron was found in the CCTalpha gene from A. rosea, and none were present in the N. gruberi sequence. However, the intron in the A. rosea CCTalpha is shared with the two R. americana strains, M. jakobiformis and Arabidopsis (intron position 10; fig. 4A ), suggesting that it may predate the divergence of plants and these protists.

It seems significant that in cases where only one or a few introns are present in the CCT genes (e.g., Acrasis, Plasmodium, Schizosaccharomyces; fig. 4A and B ) they are located near the 5' end (in CCTalpha, intron positions 1–10 are located in the first 20% of the coding sequence, whereas introns 1–5 in CCTdelta are present in the first 11% of the gene). Such a bias is also observed for the vast majority of introns in yeast (Fink 1987Citation ; Spingola et al. 1999Citation ), and for the few remaining introns in the highly reduced nucleomorph genome of the cryptomonad alga Guillardia theta (Douglas et al. 2001Citation ). In yeast, a process of incomplete reverse transcriptase–mediated intron loss has been suggested to account for such a nonrandom distribution (Fink 1987Citation ). However, it is known that at least some of these 5'-end introns play regulatory roles in gene expression (Spingola et al. 1999Citation ), suggesting that they may persist not because of incomplete reverse transcription but because of selection against intron loss. Either way, the clustering of introns near the 5'-ends of genes is consistent with loss from a more intron-rich ancestral state. On the other hand, it is also possible that such a pattern simply reflects a bias in the process of intron insertion.

Although no introns have been described in genes from the amitochondriate diplomonads and parabasalids, indirect evidence for their existence in the parabasalid T. vaginalis has come from the discovery of a homolog of PRP8, a highly conserved protein component of the spliceosome (Fast and Doolittle 1999Citation ). A gene whose product has significant similarity to PRP8 is also present in the near complete genome of the diplomonad G. lamblia (Smith et al. 1998Citation ) (identified by searching the high-throughput genome sequence database at NCBI). Taken as a whole, the data are consistent with the possibility that the intron-sparse or intron-free nature of many protist genes is a derived feature.

How intron-rich will jakobid and malawimonad nuclear genes turn out to be? At present, such a small sample size makes it impossible to predict. Nevertheless, the data presented here suggest a different picture of spliceosomal intron evolution than is often assumed, one in which intron loss has been a significant factor in shaping eukaryotic nuclear genomes.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We wish to thank B. F. Lang, S. L. Hajduk, M. Oli, A. J. Roger, R. J. Redfield, J. M. Logsdon Jr., and J. Dacks for generously providing the protist gDNAs used in this study. We also thank A. Stoltzfus for mol2con.pl, members of the Doolittle lab for discussion and comments on the manuscript, two anonymous reviewers for helpful comments, and A. Roger for assistance with ML analyses and critical review of the manuscript. For T. brucei, preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org. Preliminary sequence data for P. falciparum chromosomes 10, 11, and 13 was obtained from The Institute for Genomic Research website (www.tigr.org). Sequencing of chromosomes 10, 11, and 13 was part of the International Malaria Genome Sequencing Project and was supported by award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. This work was supported by a grant awarded to W.F.D. by the Canadian Institutes for Health Research (CIHR). J.M.A. was supported by a Doctoral Research Award from CIHR.


    Footnotes
 
Brandon Gaut, Reviewing Editor

1 Present address: Department of Botany, University of British Columbia, Vancouver, Canada Back

Keywords: jakobids eukaryotic evolution introns chaperonins CCT Back

Address for correspondence and reprints: John M. Archibald, Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4. jarch{at}interchange.ubc.ca . Back


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Accepted for publication October 26, 2001.