Evolutionary Relationships Among “Jakobid” Flagellates as Indicated by Alpha- and Beta-Tubulin Phylogenies (original) (raw)

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20 November 2000

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Virginia P. Edgcomb, Andrew J. Roger, Alastair G. B. Simpson, David T. Kysela, Mitchell L. Sogin, Evolutionary Relationships Among “Jakobid” Flagellates as Indicated by Alpha- and Beta-Tubulin Phylogenies, Molecular Biology and Evolution, Volume 18, Issue 4, April 2001, Pages 514–522, https://doi.org/10.1093/oxfordjournals.molbev.a003830
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Abstract

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the “core jakobids”), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large “plant-protist” clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera_–_R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the “jakobids” may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.

Introduction

The “jakobids” are a collection of free-living, biflagellate protists that possess a conspicuous ventral groove used for suspension feeding (O'Kelly 1993 ). Currently, this group encompasses three named groups given the rank of family: Histionidae (Histiona, Voight 1901 ; Reclinomonas, Flavin and Nerad 1993 ), Jakobidae (Jakoba, Patterson 1990 ), and Malawimonadidae (Malawimonas, O'Kelly and Nerad 1999 ). Until recently, attempts to understand the evolutionary affinities of the jakobid flagellates have relied on ultrastructural data. These data indicate that the jakobids are most similar to an interesting collection of other flagellate groups that possess suspension feeding grooves. These groups include diplomonads, retortamonads, heteroloboseids, and the recently studied taxa Trimastix and Carpediemonas (O'Kelly 1993, 1997 ; O'Kelly, Farmer, and Nerad 1999 ; O'Kelly and Nerad 1999 ; Simpson and Patterson 1999 ; Simpson, Bernard, and Patterson 2000 ). Due to their common possession of a suspension feeding groove, these organisms, together with jakobids, have been informally named the “excavate taxa” (Simpson and Patterson 1999 ). Ultrastructural data strongly suggest that most or all excavate taxa have a common excavate ancestor; that is, the excavate taxa form either a clade or a grade (Simpson and Patterson 1999 ).

Several lines of evidence argue that jakobids are among the earliest diverging of eukaryotic lineages. First, they were implicated to be key organisms in the establishment of the mitochondrial symbiosis because of their ultrastructural similarity to the retortamonads and, indirectly, diplomonads. Diplomonads and retortamonads lack mitochondria (Brugerolle 1991 ), consistent with a divergence prior to the establishment of the mitochondrial symbiosis (Cavalier-Smith 1983, 1987 ; Sleigh 1989 ; Sogin et al. 1989 ; Sogin 1991 ; Roger, Morrison, and Sogin 1999 ). This hypothesis was supported by the early divergence of the diplomonads in molecular phylogenies of SSU rDNA and several protein families (Hashimoto et al. 1994 ; Sogin et al. 1989 ). On the basis of these data, O'Kelly suggests that the ancestral mitochondrial symbiont was first engulfed by a sister taxon to the retortamonads, producing a “jakobid-like” ancestor from which all mitochondriate eukaryotes evolved (O'Kelly 1993 ). This argument has been weakened by the discovery of genes of mitochondrial origin in the diplomonad Giardia (Hashimoto et al. 1998 ; Roger et al. 1998 ; Roger 1999 ), indicating that the symbiosis took place prior to the divergence of diplomonads and parabasalids. Nevertheless, a deeply diverging position of diplomonads among eukaryotes remains a plausible scenario and implies that mitochondriate excavate taxa such as jakobids may similarly be early-diverging eukaryotes.

Second, O'Kelly (1993) noted that the jakobids encompass all three of the basic forms of mitochondrial cristae known in eukaryotes. Histionidae have tubular cristae, while Jakoba libera has flat cristae, and the sole studied member of Malawimonadidae, Malawimonas jakobiformis, has discoidal cristae (Patterson 1990 ; O'Kelly 1993 ; O'Kelly and Nerad 1999 ). Mitochondrial cristae shape has been seen as a strongly conserved character and has been used to delimit the deepest evolutionary divisions within eukaryotes (Taylor 1976 ; Sleigh 1989 ; Patterson 1994 ). Although the presence of all three forms in jakobids implies that jakobids are not monophyletic, this was also consistent with a model in which jakobid flagellates were a stem group from which all extant mitochondriate eukaryotes emerged in at least three separate radiations (O'Kelly 1993 ).

Third, and most significantly, the mitochondrial genomes of Reclinomonas americana and Jakoba libera preserve ancestral bacterial features not found in any other eukaryotes studied to date. Mitochondrial DNAs (mtDNAs) from R. americana (Lang et al. 1997 ) and J. libera (http://megasun.bch.umontreal.ca/ogmp/projects/individual.html) possess gene clusters that closely resemble ribosomal protein operons of eubacteria. They also code for RNase P RNAs with bacterial minimum consensus structures, as well as subunits of bacterial- type RNA polymerase genes that were ancestrally present in the proto-mitochondrial symbiont genome (Lang et al. 1997, 1999 ). Indeed, all other mitochondrial genomes sequenced to date encode a subset of the proteins described in jakobid mtDNAs (Burger et al. 1999 ; Gray, Burger, and Lang 1999 ). Nonetheless, jakobid mtDNAs share several unique gene arrangements with mitochondrial genomes from other eukaryotic lineages to the exclusion of all known bacterial genomes. These gene arrangements, together with the monophyly of all mtDNAs (including those of jakobids) recovered in phylogenies of single and concatenated mitochondrial gene data sets, indicate that all known mitochondria evolved from a single endosymbiosis (Gray, Burger, and Lang 1999 ; Lang et al. 1999 ). The unique retention of many ancestral alpha-proteobacterial genes in the jakobid mtDNAs is most parsimoniously explained by “jakobids” being among the earliest diverging mitochondrion-containing taxa.

For all of their potential evolutionary significance, our phylogenetic understanding of “jakobids” is incomplete. Ultrastructural data indicate that the Jakobidae (Jakoba) and the Histionidae (Reclinomonas and Histiona) are closely related but also suggest that Malawimonas may have evolved independently from within a broader radiation of excavate taxa (O'Kelly and Nerad 1999 ; Simpson and Patterson 1999 ). Although phylogenies of concatenated mitochondrial genes also indicate that J. libera and R. americana form a clade, this group is not clearly allied with Malawimonas (Burger et al. 1999 ). In light of the possibility of jakobid polyphyly, we will refer to Jakobidae and Histionidae collectively as the “core jakobids” (Simpson and Patterson 1999 ) and treat Malawimonas as a separate lineage. Our understanding of the phylogenetic placement of both groups remains hampered by the absence of nuclear gene sequences.

Over the last decade, phylogenetic reconstructions of small-subunit ribosomal RNA (SSU rRNA) gene sequences have provided a molecular phylogenetic framework for understanding the course of early eukaryotic evolution. In these phylogenies, the amitochondriate diplomonads, microsporidia, and parabasalids consistently branch before any mitochondrion-bearing lineages (Sogin 1991 ; Cavalier-Smith 1993 ; Leipe et al. 1993 ). However, there is doubt as to whether SSU rRNA correctly identifies the deepest diverging groups and their relative branching order (Leipe et al. 1993 ; Hirt et al. 1999 ). Large disparities in the rates of evolution (leading to long-branch attraction) and extreme biases in base compositions of different SSU rRNA sequences, especially among “early-branching” eukaryotic taxa, may confound accurate phylogenetic reconstruction (Loomis and Smith 1990 ; Hasegawa et al. 1993 ; Hasegawa and Hashimoto 1993 ; Galtier and Gouy 1995 ; Roger et al. 1999 ; Stiller and Hall 1999 ; Philippe and Germot 2000 ). Highly conserved protein-coding genes might be less sensitive to some of these problems for several of the taxa identified as “deep-branches” in SSU rRNA trees (Hasegawa et al. 1993 ; Hashimoto et al. 1994 ; Roger et al. 1996 ; Hirt et al. 1999 ). Therefore, the development of these markers in combination with SSU rRNA should help in determining a more robust global eukaryotic phylogeny.

To this end, we focused on the tubulin gene family as a phylogenetic marker for early eukaryotic evolution. This gene family consists of three highly conserved subfamilies, alpha-, beta-, and gamma-tubulin, that arose from a series of gene duplications in early eukaryotic evolution (Edlind et al. 1996 ; Keeling and Doolittle 1996 ). Currently, alpha- and beta-tubulin genes have the widest taxonomic representation and have been identified in the putatively early diverging eukaryotic lineages (Kirk-Mason, Turner, and Chakraborty 1988 ; Burns 1991 ; Katiyar and Edlind 1994, 1996 ; Edlind et al. 1996 ; Keeling and Doolittle 1996 ). In order to study the relationships among the “jakobid” flagellates and their relationship to other “excavate” protist lineages, we characterized the alpha- and beta-tubulin genes from four jakobid species: Jakoba libera, Jakoba incarcerata, Reclinomonas americana, and Malawimonas jakobiformis.

Materials and Methods

Sources of DNA

Genomic DNA from J. libera (ATCC 50422), M. jakobiformis (ATCC 50310), and R. americana (ATCC 50284) were kindly provided by Dr. B. Franz Lang, Université de Montreal, Canada. Genomic DNA from R. americana (ATCC 50283) was provided by Tom Nerad, American Type Culture Collection. Genomic DNA was extracted from the “type culture line” of J. incarcerata (Bernard, Simpson, and Patterson 2000 ) using the Puregene DNA Isolation Kit (Gentra Systems).

PCR Amplification, Cloning, and Sequencing

Alpha- and beta-tubulin genes were amplified from genomic DNA. Degenerate primers were previously designed based on highly conserved N- and C-terminal regions of the alpha- and beta-tubulin gene families (Roger 1996 ). BtubA was based on the amino acid motif GQCGNQ and had the sequence 5′-GCAGGNCARTGYGGNAAYCA-3′; BtubB was based on MDEMEFT and had the sequence 5′-AGTRAAYTCCATYTCRTCCAT-3′; AtubA was based on QVGNCWE with the sequence 5′-RGTNGGNAAYGCNTGYTGGGA-3′; and AtubB was based on WYVGEGM with the sequence 5′-CCATNCCYTCNCCNACRTACCA-3′. These primer combinations amplified >90% of the coding region of each gene. Amplification reactions were performed with 50–100 ng of total genomic DNA under standard conditions both in the presence and in the absence of 5%–6% acetamide (final concentration). Thermal cycling consisted of 35 cycles with an annealing temperature of 48–55°C for 1 min, an extension temperature of 72°C for 1 min, and a denaturing temperature of 94°C for 30 s.

PCR products were purified from agarose gels using the Prep-A-Gene DNA Purification Kit (BioRad) and cloned directly into pGEM-T-easy vector (Promega) or TOPO-XL vector (Invitrogen, Inc.). Sequence data were gathered on a LI-COR 4200L automated sequencing apparatus using infrared dye-labeled T7 and M13R primers (LI-COR) and a cycle sequencing protocol (Sequitherm, Epicenter, Inc.). Single-stranded sequence of 4–10 clones of each PCR product was initially determined to test for the presence of multiple distinct copies of tubulin genes within each organism and to determine internal restriction sites for subcloning. Gene fragments of 100–900 bp were generated by restriction enzyme digestion of plasmid templates and subsequently subcloned into pBluescript KS (−) or pUC-19 vectors. Full- length double-stranded DNA sequences of subcloned distinct gene copies were determined by combined analysis of both complete gene and subcloned template data using ALIGNIR sequence assembly software (LI-COR).

Phylogenetic Analysis

Nucleotide sequence data were submitted to NCBI BLAST (Basic Local Alignment Search Tool) (Altschul et al. 1990 ) for comparison with available sequence databases to verify tubulin gene identity and locate introns. Protein sequences were compiled and manually aligned in PAUP*, version 4.0b2 (Swofford 1999 ), along with tubulin sequences from representatives of diverse taxa obtained using the NCBI Entrez protein sequence search program. In total, a data set of 63 alpha tubulins and 85 beta tubulin sequences was compiled, with representatives from all available eukaryotic groups.

The alignment of intron sequences was obtained using the CLUSTAL W program, version 1.7 (Thompson, Higgins, and Gibson 1994 ) under default parameter settings and manually adjusted by eye. Phylogenetic analyses of the R. americana complete beta-tubulin DNA sequences including introns were performed using the maximum-likelihood (ML) method implemented in PUZZLE, version 4.02 (Strimmer and von Haeseler 1996 ), employing the Hasegawa, Kishino and Yano (1985) model with an eight-category discrete gamma model of among-site rate variation (HKY+Γ). ML estimation on a neighbor-joining topology was used to derive a transition : transversion parameter of 1.21 and a gamma shape parameter (α) of 0.02. Bootstrapping (1,000 resamplings) was accomplished using the SEQBOOT and CONSENSE programs (from PHYLIP [Felsenstein 1995] ) in combination with QBOOT, a unix shell-script that allows quartet ML bootstrap analysis with PUZZLE, version 4.02 (available on request from A.J.R.).

Phylogenetic analyses of the amino acid sequences were performed using both the protein ML distance (ML-distance) and the ML quartet puzzling (ML-QP) methods implemented in PUZZLE, version 4.02 (Strimmer and von Haeseler 1996 ). Data missing in more than a single sequence and regions of ambiguous alignment were removed, yielding 381 sites for the alpha-tubulin analysis, 387 sites for the beta-tubulin analysis, and 768 sites for a combined (concatenated) alpha- plus beta- tubulin analysis. The ML model employed in both distance and likelihood analyses was the Jones, Taylor, and Thornton (1992) amino acid replacement matrix with an eight-category discrete gamma model of among-sites rate variation (JTT+Γ). The gamma shape parameter α was estimated by ML optimization on a neighbor-joining topology to be 0.72 for the alpha-tubulin data set, 0.52 for the beta-tubulin data set, and 0.57 for the concatenated alpha+beta-tubulin data set. Phylogenetic trees were inferred from the ML distances using the Fitch-Margoliash algorithm with global rearrangements (FITCH, version 3.57c [Felsenstein 1995] ). ML distance bootstrap values for bipartitions were calculated by analysis of 100 resampled data sets generated with the SEQBOOT program and analyzed with the PUZZLEBOOT script (http://www.tree-puzzle.de/) in conjunction with PUZZLE, version 4.02, and CONSENSE. Full protein ML analyses were performed using the quartet puzzling algorithm in PUZZLE, version 4.02, utilizing 1,000 puzzling steps.

Results and Discussion

Tubulin Genes from Jakobids

Amplification reactions for alpha-tubulin genes for J. incarcerata, J. libera, R. americana 50283, and M. jakobiformis all yielded a product of roughly the expected size of an alpha-tubulin gene without introns. A single product was also obtained for the beta-tubulin gene of J. incarcerata. Two distinct beta-tubulin gene products were obtained from R. americana 50283 (99.1% identical at the nucleotide level), R. americana 50284 (99.6% identity), and M. jakobiformis (98.4% identity). No introns were found in alpha- or beta-tubulins of M. jakobiformis and J. incarcerata or in the alpha-tubulin genes of R. americana and J. libera. However, the two R. americana 50283 beta tubulin paralogs contained introns of 70 (clone 22) and 78 (clone 18) bp in length, while the R. americana 50284 paralogs contained introns of 70 (clone 14) and 75 (clone 12) bp in length, located in the same position, starting at the 56th base of the sequences (located between a threonine and a glycine codon). Phylogenetic analysis of these tubulin paralogs revealed that the gene duplication that created the two copies of beta-tubulin in R. americana appears to have predated the divergence between strains 50283 and 50284 (data not shown). Similarly, initial sequences from six J. libera beta-tubulin clones revealed three distinct gene copies that were distinguishable by intron sequences of 80 (clone 6), 134 (clone 1), and 157 (clone 2) bp in length, starting at the 116th base of the beta- tubulin genes (located between a glutamate and an alanine codon).

Another noteworthy property of the tubulin genes described here is a moderate to extreme codon usage bias toward codons ending in G or C (table 1 ). The GC- bias of alpha- and beta-tubulin genes from each organism were very similar, confirming that each set of genes derive from the same genome.

Tubulin Phylogeny

The inferred amino acid sequences for alpha-tubulin genes from J. libera, J. incarcerata, R. americana 50283, and M. jakobiformis, as well as the beta-tubulin genes from these organisms, plus R. americana 50284, were added to our alignments of alpha-tubulin and beta- tubulin sequences. Preliminary phylogenetic analyses using all sequences in our alignment were performed. From these analyses, we identified and selectively removed partial sequences and sequences that were extremely divergent paralogs or orthologs (to avoid problems with long-branch attraction artifacts) and pared down representation of major eukaryotic groups to yield final alignments of 42 alpha-tubulin and 39 beta-tubulin sequences that were amenable to more rigorous phylogenetic analyses. Separate analyses were performed on the alpha- and beta-tubulin (fig. 1 ) data sets and for a combined alpha- and beta-tubulin data set that included organisms for which both sequences (or for which related sequences) were represented (fig. 2 ). We did not attempt to reciprocally root the alpha- and beta-tubulin trees on each other. The two families are extremely divergent from one another, and previous analyses showed that when used as reciprocal outgroups, they artifactually attract fast-evolving sequences to the base of each of the paralog subtrees (Keeling, Deane, and McFadden 1998 ; Keeling et al. 1999 ). Instead, sequences from diplomonads were arbitrarily drawn as the outgroup for each of the three phylogenies, as these amitochondriate protists consistently occupy basal positions in rooted trees based on ribosomal RNA, EF-1α, EF-2, and RPB1 (Sogin 1991 ; Leipe et al. 1993 ; Hashimoto et al. 1994 ; Hirt et al. 1999 ).

A variety of traditional eukaryotic groups that are well established on the basis of morphology or molecular phylogeny are also recovered in either alpha- or beta-tubulin trees (Edlind et al. 1996 ; Keeling and Doolittle 1996 ) (figs. 1 and 2 ). In addition, several “higher- level” eukaryotic groupings have been observed, including fungi plus microsporidia, animals plus fungi (this clade is likely to include taxa which are neither animals nor fungi, e.g., choanoflagellates), and ciliates plus apicomplexa (alveolates). Phylogenies based on SSU rRNA, alpha-tubulin, and beta-tubulin support the animals-plus-fungi assemblage (represented in this data set by metazoa, fungi, and microsporidia) and the grouping of the alveolates (Sogin 1991 ; Wainright et al. 1993 ; Edlind et al. 1996 ; Keeling and Doolittle 1996 ; Keeling, Deane, and McFadden 1998 ). In addition, elongation factors and actins demonstrate a common evolutionary history of animals plus fungi exclusive of most other eukaryotes (Baldauf and Palmer 1993 ). However, alpha- and beta-tubulin phylogenies also display attributes not always supported by other gene-based trees (Edlind et al. 1996 ; Keeling et al. 1999 ). In agreement with other studies (Edlind et al. 1996 ; Keeling et al. 1999 ), our analyses of single tubulin data sets show that microsporidia group strongly with the fungi (figs. 1 and 2 ). This is in contrast to their deep placement in SSU rRNA trees (Leipe et al. 1993 ) but in agreement with other molecular phylogenies, including mitochondrial HSP70 (Germot, Philippe, and Le Guyader 1997 ; Hirt et al. 1997 ), TATA-box binding protein (Fast, Logsdon, and Doolittle 1999 ), elongation factor-2, RPB1 (Hirt et al. 1999 ), ValtRS (Weiss et al. 1999 ), and, most recently, large-subunit ribosomal RNA (LSU rRNA) (Van de Peer, Ben Ali, and Meyer 2000 ). Also unlike rRNA trees, tubulin phylogenies do not show many independent deep-branching lineages leading to a “crown” radiation of eukaryotes if diplomonads are considered the outgroup. Instead, a deep split occurs within eukaryotes separating two robust groupings, a plant-protist “superclade” and an animals-plus-fungi group, with several protist lineages intervening (fig. 1 ). Although the exact composition of the plant-protist “superclade” varies between our alpha- and beta-tubulin phylogenies and other published analyses, the group is consistently composed of Viridiplantae (land plants and chlorophyte algae), stramenopiles, cercozoans, cryptomonads, Euglenozoa, and heteroloboseids (fig. 1 ) (Edlind et al. 1996 ; Keeling, Deane, and McFadden 1998 ; Keeling et al. 1999 ). Intervening taxa between the two major clades include Eumycetozoa, parabasalids, and diplomonads. The Eumycetozoa weakly associate with animals plus fungi in beta-tubulin analysis; however, in both alpha-tubulin and combined alpha- plus beta-tubulin phylogenies, they strongly associate with the plant protist superclade (figs. 1 and 2 ). Similarly, parabasalids and diplomonads are sister taxa in beta-tubulin phylogenies (fig. 1_B_ ), yet parabasalids, not diplomonads, show some affinity to the animals-plus-fungi group in alpha-tubulin trees (fig. 1_A_ ).

Placement of “Jakobid” Flagellates in Tubulin Phylogenies

ML distance/Fitch and ML-QP trees for each of the three analyses (figs. 1_A,_ 1_B,_ and 2 ) show that the “jakobids” in the broad sense (Reclinomonas, Jakoba, and Malawimonas) (O'Kelly 1993 ) and the “excavate taxa” (in this analysis, jakobids, heteroloboseids, and diplomonads) do not form clades. However, our analyses do show a close grouping between beta-tubulins from J. libera, R. americana 50283, and R. americana 50284 (99% bootstrap support in a distance analysis, and quartet puzzling support of 83%) (fig. 2_B_ ). The extremely close relationship between R. americana and J. libera beta-tubulins (fig. 1_B_ ) is particularly interesting because these two sequences each harbor a single nonhomologous intron that occupies different positions in the gene (data not shown). Neither intron position is found in any other beta-tubulin gene studied to date (J. M. Logsdon Jr., personal communication), thus indicating recent intron turnover in beta-tubulin in the R. americana_–_J. libera lineage.

Interestingly, the J. libera_–_R. americana grouping is not recovered in alpha-tubulin phylogenies by either method of analysis (fig. 1_A_ ). Lack of support for this grouping, which is well supported by other data (discussed below), is puzzling and may have several causes. For instance, placement of R. americana and J. libera alpha-tubulin sequences may be obscured by the overall poorer support for monophyletic groupings observed in the alpha-tubulin tree, especially within the plant-protist superclade (fig. 1_A_ ). It is also possible that one of these sequences represents an aberrant paralog of alpha-tubulin which could have life-cycle-specific expression and has an altered pattern of substitution, a frequently observed phenomenon in tubulins (Kube-Granderath and Schliwa 1998 ). If so, then the orthologous alpha- tubulin gene(s) from one or another of these organisms may not have been detected in our PCR approach, perhaps due to introns interrupting the primer binding sites in the genes. However, the J. libera_–_R. americana relationship was well supported in the combined alpha- and beta-tubulin analysis (91% bootstrap support and 90% QP support; fig. 2 ), indicating that the alpha-tubulin data do not significantly conflict with the beta- tubulin data, but instead lack strong enough phylogenetic signal to resolve the relationships between these taxa.

Also unexpected was the positioning of J. incarcerata in tubulin trees. Jakoba incarcerata was assigned to Jakoba primarily on the basis of light microscope observations (Bernard, Simpson, and Patterson 2000 ). Ultrastructural data support assignation of J. incarcerata to the “core jakobids” (Bernard, Simpson, and Patterson 2000 ; unpublished data) but also suggest that J. incarcerata may be basal to other known “core jakobids” (i.e., Jakoba may not be monophyletic). Jakoba incarcerata would therefore be expected to group with J. libera and R. americana in a “core jakobid” clade (Simpson and Patterson 1999 ), but not necessarily to group specifically with J. libera. However, in our beta-tubulin analyses, J. incarcerata did not cluster with these taxa, emerging separately within the plant-protist superclade in beta-tubulin trees. In alpha-tubulin phylogeny, it joined the animals-plus-fungi–parabasalid grouping with very weak support (45% distance bootstrap and 59% QP support; fig. 1_A_ ). Interestingly, this sequence robustly grouped with the plant-protist superclade in the combined alpha- plus beta-tubulin analysis (100% distance bootstrap and 74% QP support) and emerged with moderate support as the earliest-diverging lineage within this clade (70% distance bootstrap and QP support). Similarly, M. jakobiformis occupied a relatively basal position in the plant-protist superclade in all three analyses, varying in position only slightly (figs. 1 and 2 ).

To evaluate the stability of the position of J. incarcerata in the combined analysis in light of the weakly supported affinity of J. incarcerata for the animals- plus-fungi–parabasalid grouping in the alpha-tubulin analysis, we performed a separate combined alpha- plus beta-tubulin analysis that included the alpha- and beta- tubulin genes from a second diplomonad, Giardia intestinalis, and a composite trichomonad sequence composed of Trichomonas vaginalis beta-tubulin and Monocercomonas sp. alpha-tubulin (data not shown). In this analysis, there were only minor changes in support values at several nodes, and the position of the jakobids, including J. incarcerata, did not change from the combined alpha- plus beta-tubulin analysis presented here.

There is little, if any, resolution in the branching order among the major groups in the plant-protist superclade in our tubulin trees, and, in general, the jakobid lineages show inconsistent affinities to other taxa in the various analyses. One exception is the tendency in beta- tubulin trees for the J. libera_–_R. americana group to branch with the heteroloboseid Naegleria and the euglenozoan Euglena (fig. 1_B_ ). This tendency is amplified, albeit weakly (44% distance bootstrap support and 56% QP support), in the combined alpha- plus beta-tubulin analysis, in which the J. libera_–_R. americana group became a sister group of a Heterolobosea-Euglenozoa clade. The best resolution of the major phyla and their relative branching order was obtained in our combined alpha- plus beta-tubulin analyses, yet support values for some groups (e.g., Viridiplantae) remained low. It is likely that the extreme conservation of both tubulin genes leave few phylogenetically informative sites, especially for groups in the plant-protist superclade. In this respect, ancient phylogenetic signal appears to be retained in tubulin sequences, but its overall amount is low.

Jakobid Polyphyly

Our observation of a J. libera_–_R. americana grouping is in agreement with concatenated mitochondrial gene phylogenies (Burger et al. 1999 ; Gray, Burger, and Lang 1999 ; Burger et al. 2000 ), SSU rRNA phylogenies (unpublished data), and shared unique mtDNA gene composition between these taxa (e.g., their common possession of bacterial DNA-dependent RNA polymerase genes) (Lang et al. 1999 ). Concatenated mitochondrial gene phylogenies also support the distinctness of Malawimonas from “core jakobids” and show them emerging from within a strikingly similar plant-protist superclade (although the monophyly of this clade is not well supported). These results are consistent with recent evaluations of ultrastructural features of excavate taxa (O'Kelly, Farmer, and Nerad 1999 ; O'Kelly and Nerad 1999 ; unpublished data) which indicate that “core jakobids” (including J. libera and R. americana) share several characters not found in other excavate taxa but that only one or two features of debatable importance are unique to Malawimonas and “core jakobids.” Given the conflict between ultrastructural and tubulin data in the placement of J. incarcerata, further data on this taxon would be desirable. Unfortunately, mitochondrial or nuclear gene data have not yet been published.

The Cristal Hypothesis

O'Kelly's (1993) original suggestion for jakobids as a stem group from which all three basic cristal morphologies arose requires the jakobids to be paraphyletic, giving rise to at least three clades of nonjakobid organisms. While it could be argued that this degree of paraphyly of jakobids is reconcilable with our results, it appears safe to reject O'Kelly's hypothesis from the evidence at hand. First, O'Kelly's hypothesis requires that the J. libera_–_R. americana grouping be interrupted by at least two nonjakobid clades (one essentially platycristate, the other tubulocristate). Second, it would predict that Malawimonas would fall adjacent to Heterolobosea and Euglenozoa to form a clade of discicristate organisms. In fact, it is the J. libera_–_R. americana clade which shows some affinity for Heterolobosea and Euglenozoa (figs. 1 and 2 ).

Jakobids and Deep-Branching Excavate Taxa

Tubulin genes are the first molecules to be used to evaluate the affinities of jakobids to amitochondriate “excavate” protist lineages. We did not observe a close relationship between the diplomonads and any of the jakobid lineages in this study, although sequences from Malawimonas and J. incarcerata branched near the base of the plant-protist superclade and therefore emerged in a region of the tree close to the diplomonad branch. Currently, the “excavate hypothesis” can be rationalized in terms of tubulin phylogenies in the following manner. If some jakobid lineages (e.g., J. incarcerata and Malawimonas) are among the deepest branches in a plant- protist superclade, and diplomonads are the outgroup to these and all other eukaryotes, then the common ancestor of the plant-protist superclade and the common ancestor of extant eukaryotes could have had an “excavate” phenotype. Paradoxically, it is precisely R. americana and J. libera that display the most primitive mitochondrial genome organization (Lang et al. 1999 ), yet they do not seem to occupy a particularly deep branch in tubulin trees. Clearly, more molecular data will be required from jakobids and other excavates, such as retortamonads, Trimastix, and Carpediemonas, to decide where these protists fit in the large-scale phylogeny of eukaryotes.

Perhaps even more problematic will be understanding the basis of conflicts between large-scale eukaryotic phylogenies inferred with the various nuclear gene markers (e.g., tubulin vs. SSU rRNA phylogenies). Substitution rate calibration analyses of “crown” eukaryotic rRNAs support the division between an animals-plus- fungi group and a large heterogeneous plant-protist clade of eukaryotes (Van de Peer et al. 1996 ), as do concatenated mitochondrial genes (Burger et al. 1999 ). However, the most conspicuous disagreement between SSU rRNA trees and tubulin trees is the fact that “deep” lineages in SSU rRNA trees (Leipe et al. 1993 ), such as Microsporidia, Eumycetozoa, Euglenozoa, and Heterolobosea, emerge from within the these two major eukaryotic clades in tubulin trees. Several authors have suggested that the deep-branching position of these and other taxa within SSU rRNA trees could result from long-branch attraction artifacts (Embley and Hirt 1998 ; Keeling, Deane, and McFadden 1998 ; Philippe and Adoutte 1998 ; Roger, Morrison, and Sogin 1999 ) and that the alternative positioning of these taxa within the tubulin tree may be correct (Embley and Hirt 1998 ; Keeling, Deane, and McFadden 1998 ; Dacks and Roger 1999 ). Although this seems likely to be the case for Microsporidia (Keeling and McFadden 1998 ), we should also be cautious in placing too much confidence in tubulin phylogenies. Most taxa that lack flagella or centrioles, such as Microsporidia, higher fungi, Entamoeba, Dictyostelium, and red algae, tend to form extremely long branches in tubulin trees, and often group together (Edlind et al. 1996 ; Keeling and Doolittle 1996 ; Keeling et al. 1999 ; Keeling, Luker, and Palmer 2000 ; unpublished data), as do aberrant developmentally regulated tubulin isoforms (data not shown). Therefore, much of the structure of tubulin-based trees could also result from long-branch attraction effects.

Supplementary Material

GenBank accession numbers for newly deposited sequences are as follows: J. incarcerata clone 22 alpha- tubulin, AF267179; J. libera alpha-tubulin, AF267180; M. jakobiformis alpha-tubulin, AF267181; R. americana sp. clone 44 alpha-tubulin, AF267182; J. incarcerata beta-tubulin, AF267183; J. libera clone 3 beta-tubulin, AF267184; M. jakobiformis clone 1 beta-tubulin, AF267185; M. jakobiformis clone 7 beta-tubulin, AF267186; R. americana 50283 clone 22 beta-tubulin, AF267190; R. americana 50283 clone 18 beta-tubulin, AF267189; R. americana 50284 clone 12 beta-tubulin, AF267187; R. americana 50284 clone 14 beta-tubulin, AF267188.

B. Franz Lang, Reviewing Editor

1

Keywords: jakobid alpha-tubulin beta-tubulin phylogeny flagellates

1

Present address: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

3

Address for correspondence and reprints: Mitchell L. Sogin, Josephine Bay-Paul Center of Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543. sogin@evol5.mbl.edu.

Table 1 Codon Usage Bias in Jakobid Flagellates

Table 1 Codon Usage Bias in Jakobid Flagellates

Fig. 1.—Phylogeny of eukaryotes inferred from (A) alpha-tubulin and (B) beta-tubulin protein sequences. Phylogenetic trees were inferred with the Fitch-Margoliash algorithm from maximum-likelihood distance matrices calculated with the JTT+Γ model of amino acid replacement. The gamma distribution shape parameter alpha was 0.72 for the alpha-tubulin analysis (A) and 0.52 for the beta-tubulin analysis (B). Support values shown near branches were computed using ML distance/Fitch-Margoliash bootstrap analysis (100 replicates) and ML quartet puzzling (1,000 puzzling steps) and are displayed in that order. Jakobid sequences (determined in this study) are highlighted and identified by the letters “A” (alpha-tubulin) and “B” (beta-tubulin) and the number of the clone used for the analysis. In cases in which there were significant (more than six amino acids) differences between clones sequenced, more than one clone is included in the analysis. The scale bar indicates expected sequence divergence per unit branch length, expressed as substitutions per site. The question mark indicates the uncertain origin of the beta-tubulin sequence ascribed to the rhodophyte Porphyra purpurea. This sequence appears to branch within the stramenopiles, and its identity is therefore questionable (see also Keeling et al. [1999] for discussion of this point)

Fig. 2.—Phylogeny of eukaryotes inferred from concatenated alpha+beta-tubulin protein sequences. The tree topology was inferred with the Fitch-Margoliash algorithm from a maximum-likelihood (ML) distance matrix calculated with the JTT+Γ model of amino acid replacement. The gamma distribution shape parameter alpha was 0.57. Support values shown near branches were computed using ML distance/Fitch-Margoliash distance bootstrap analysis (100 replicates) and ML quartet puzzling (1,000 puzzling steps) and are displayed in that order. Jakobid sequences are highlighted, and the genes are identified by the letters “A” (alpha-tubulin) and “B” (beta-tubulin) and the number of the clone used for the combined analysis in each case. The scale bar indicates expected sequence divergence per unit branch length, expressed as substitutions per site

We thank John M. Archibald and Joel Dacks for helpful comments on early drafts of the manuscript; B. Franz Lang, G. Burger, and C. O'Kelly for kindly providing genomic DNAs; and John M. Logsdon Jr. for the tubulin intron information. This work was supported by NASA grant NAG5-4895 to M.L.S., NASA Astrobiology Cooperative Agreement NCC2-1054, and continuing support from the Unger G. Vetlesen Foundation to M.L.S. V.P.E. and A.J.R. both contributed equally to this work.

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