Multigene phylogenies of diverse Carpediemonas-like organisms identify the closest relatives of 'amitochondriate' diplomonads and retortamonads

Diplomonads, retortamonads, and "Carpediemonas-like" organisms (CLOs) are a monophyletic group of protists that are microaerophilic/anaerobic and lack typical mitochondria. Most diplomonads and retortamonads are parasites, and the pathogen Giardia intestinalis is known to possess reduced mitochondrion-related organelles (mitosomes) that do not synthesize ATP. By contrast, free-living CLOs have larger organelles that superficially resemble some hydrogenosomes, organelles that in other protists are known to synthesize ATP anaerobically. This group represents an excellent system for studying the evolution of parasitism and anaerobic, mitochondrion-related organelles. Understanding these evolutionary transitions requires a well-resolved phylogeny of diplomonads, retortamonads and CLOs. Unfortunately, until now the deep relationships amongst these taxa were unresolved due to limited data for almost all of the CLO lineages. To address this, we assembled a dataset of up to six protein-coding genes that includes representatives from all six CLO lineages, and complements existing rRNA datasets. Multigene phylogenetic analyses place CLOs as well as the retortamonad Chilomastix as a paraphyletic basal assemblage to the lineage comprising diplomonads and the retortamonad Retortamonas. In particular, the CLO Dysnectes was shown to be the closest relative of the diplomonads + Retortamonas clade, with strong support. This phylogeny is consistent with a drastic degeneration of mitochondrion-related organelles during the evolution from a free-living organism resembling extant CLOs to a probable parasite/commensal common ancestor of diplomonads and Retortamonas.     

Evolutionary history of "early-diverging" eukaryotes: the excavate taxon Carpediemonas is a close relative of Giardia

Diplomonads, such as Giardia, and their close relatives retortamonads have been proposed as early-branching eukaryotes that diverged before the acquisition-retention of mitochondria, and they have become key organisms in attempts to understand the evolution of eukaryotic cells. In this phylogenetic study we focus on a series of eukaryotes suggested to be relatives of diplomonads on morphological grounds, the "excavate taxa". Phylogenies of small subunit ribosomal RNA (SSU rRNA) genes, alpha-tubulin, beta-tubulin, and combined alpha- + beta-tubulin all scatter the various excavate taxa across the diversity of eukaryotes. But all phylogenies place the excavate taxon Carpediemonas as the closest relative of diplomonads (and, where data are available, retortamonads). This novel relationship is recovered across phylogenetic methods and across various taxon-deletion experiments. Statistical support is strongest under maximum-likelihood (ML) (when among-site rate variation is modeled) and when the most divergent diplomonad sequences are excluded, suggesting a true relationship rather than an artifact of long-branch attraction. When all diplomonads are excluded, our ML SSU rRNA tree actually places retortamonads and Carpediemonas away from the base of the eukaryotes. The branches separating excavate taxa are mostly not well supported (especially in analyses of SSU rRNA data). Statistical tests of the SSU rRNA data, including an "expected likelihood weights" approach, do not reject trees where excavate taxa are constrained to be a clade (with or without parabasalids and Euglenozoa). Although diplomonads and retortamonads lack any mitochondria-like organelle, Carpediemonas contains double membrane-bounded structures physically resembling hydrogenosomes. The phylogenetic position of Carpediemonas suggests that it will be valuable in interpreting the evolutionary significance of many molecular and cellular peculiarities of diplomonads.     

Phylogenetic affinity of a Giardia lamblia cysteine desulfurase conforms to canonical pattern of mitochondrial ancestry

Among a few potential archezoan groups, only the Metamonada (diplomonads, retortamonads, and oxymonads) still retain the status of amitochondriate protists that diverged before the acquisition or retention of mitochondria. Indeed, finding that diplomonad genomes harbor a gene encoding a mitochondrial type chaperonin 60, the most compelling evidence for their secondarily amitochondriate nature, may be interpreted as an acquisition of this important general chaperone during some transient alpha-proteobacterial endosymbiosis. Recently published data on the cysteine desulfurase IscS demonstrated an alpha-proteobacterial origin of mitochondrial enzymes including a diplomonad Giardia lamblia homolog. An extended phylogenetic analysis of IscS is reported here that revealed a full canonical pattern of mitochondrial ancestry for the giardial enzyme. The above canonical pattern, a sister group relationship of mitochondria and rickettsiae exclusive of free-living alpha-proteobacteria, was robustly confirmed by a comprehensive analysis of Cob and Cox1 subunits of the respiratory chain encoded by resident mitochondrial genes. Given that Fe-S cluster assembly involving IscS represents an essential mitochondrial function, these data strongly suggest that diplomonads once harbored bona fide mitochondria.     

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.     

A wide diversity of previously undetected free‐living relatives of diplomonads isolated from marine/saline habitats

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free‐living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas‐like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small‐subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad‐retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation – it is likely that undersampling at the level of major lineages is still widespread for protists.     

Lateral transfer of the gene for a widely used marker, alpha-tubulin, indicated by a multi-protein study of the phylogenetic position of Andalucia (Excavata)

alpha-Tubulin is one of the most widely used markers for estimating deep-level phylogenetic relationships amongst eukaryotes. We sequenced 6-7 nuclear protein-coding genes, including alpha-tubulin, from the two described species of the enigmatic jakobid(-like) excavate protist Andalucia. Concatenated protein phylogenies place Andalucia in a clade with other jakobids, Euglenozoa and Heterolobosea. Individual gene trees, except that of alpha-tubulin, do not conflict strongly with this position. In alpha-tubulin trees, Andalucia instead falls in a strongly supported clade with diplomonads, parabasalids and opisthokonts (including animals and fungi), and branches with diplomonads. This clade is robust to changes in taxon sampling, and is unlikely to represent long-branch attraction, compositional heterogeneity artefact, or segmental gene conversion. Phylogenies estimated without alpha-tubulin strongly support the original position for Andalucia, and also reinforce recent studies in placing diplomonads and parabasalids with Preaxostyla, not opisthokonts. alpha-Tubulin seems to have experienced two or more eukaryote-to-eukaryote lateral gene transfer (LGT) events, one perhaps from an ancestral opisthokont to an ancestor of diplomonads and parabasalids, or vice versa, and one probably from the diplomonad lineage to Andalucia. Like EF-1alpha/EFL, alpha-tubulin has a complex history that needs to be taken into account when using this marker for deep-level phylogenetic inference.     

The phylogenetic position of the pelobiont Mastigamoeba balamuthi based on sequences of rDNA and translation elongation factors EF-1alpha and EF-2

The taxonomic position and phylogenetic relationships of the Pelobionta, an amitochondriate amoeboflagellate group, are not yet completely settled. To provide more information, we obtained sequences for the large subunit rDNA gene, the gene for translation elongation factor 1alpha, and for a large part of the gene encoding translation elongation factor 2 from a representative of this group, Mastigamoeba balamuthi (formerly Phreatamoeba balamuthi). The gene for the large subunit rDNA was unusually large compared to those of other protists, a phenomenon that had previously been observed for the gene encoding the small subunit rDNA. Phylogenetic reconstruction using a maximum likelihood method was performed with these sequences, as well as the gene encoding the small subunit rDNA. When evaluated individually, the M. balamuthi genes for the small and large subunit rDNAs and elongation factor 1alpha had a most recent common ancestor with either the Mycetozoa (slime molds) or with Entamoeba histolytica. A clade formed by M. balamuthi, E. histolytica, and Mycetozoa was not rejected statistically for any of the sequences. A combined maximum likelihood analysis using 3,935 positions from all molecules suggested that these three taxonomic units form a robust clade. We were unable to resolve the closest group to this clade using the combined analysis. These findings support the notion, which had previously been proposed primarily on cytological evidence, that both M. balamuthi and E. histolytica are closely related to the Mycetozoa and that these three together represent a major eukaryotic lineage.     

Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies

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.     

Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA,alpha-tubulin and HSP90 genes: Implications for the evolutionary history of the double karyomastigont of diplomonads

Background  

Fornicata is a relatively recently established group of protists that includes the diplokaryotic diplomonads (which have two similar nuclei per cell), and the monokaryotic enteromonads, retortamonads and Carpediemonas, with the more typical one nucleus per cell. The monophyly of the group was confirmed by molecular phylogenetic studies, but neither the internal phylogeny nor its position on the eukaryotic tree has been clearly resolved.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their ‘lacking mitochondria’ and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplo-monads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Primary Structure and Phylogenetic Relationships of a Malate Dehydrogenase Gene from Giardia lamblia

The lactate and malate dehydrogenases comprise a complex protein superfamily with multiple enzyme homologues found in eubacteria, archaebacteria, and eukaryotes. In this study we describe the sequence and phylogenetic relationships of a malate dehydrogenase (MDH) gene from the amitochondriate diplomonad protist, Giardia lamblia. Parsimony, distance, and maximum-likelihood analyses of the MDH protein family solidly position G. lamblia MDH within a eukaryote cytosolic MDH clade, to the exclusion of chloroplast, mitochondrial, and peroxisomal homologues. Furthermore, G. lamblia MDH is specifically related to a homologue from Trichomonas vaginalis. This MDH topology, together with published phylogenetic analyses of β-tubulin, chaperonin 60, valyl-tRNA synthetase, and EF-1α, suggests a sister-group relationship between diplomonads and parabasalids. Since these amitochondriate lineages contain genes encoding proteins which are characteristic of mitochondria and α-proteobacteria, their shared ancestry suggests that mitochondrial properties were lost in the common ancestor of both groups. Received: 14 September 1998 / Accepted: 29 December 1998     

Phylogenetic positions of several amitochondriate protozoa-Evidence from phylogenetic analysis of DNA topoisomerase II

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Inference of the phylogenetic position of oxymonads based on nine genes: support for metamonada and excavata

Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most challenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding groove that is proposed to be homologous. Euglenozoans, parabasalids, and oxymonads have also been associated with Excavata as their relationships to one or more core excavate taxa were demonstrated. However, the main barrier to the general acceptance of Excavata is that its existence is founded primarily on cytoskeletal similarities, without consistent support from molecular phylogenetics. In gene trees, Excavata are typically not recovered together. In this paper, we present an analysis of the phylogenetic position of oxymonads (genus Monocercomonoides) based on concatenation of eight protein sequences (alpha-tubulin, beta-tubulin, gamma-tubulin, EF-1alpha, EF-2, cytosolic (cyt) HSP70, HSP90, and ubiquitin) and 18S rRNA. We demonstrate that the genes are in conflict regarding the position of oxymonads. Concatenation of alpha- and beta-tubulin placed oxymonads in the plant-chromist part of the tree, while the concatenation of other genes recovered a well-supported group of Metamonada (oxymonads, diplomonads, and parabasalids) that branched weakly with euglenozoans--connecting all four excavates included in the analyses and thus providing conditional support for the existence of Excavata.     

The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1-V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba, Endolimax nana, and Phreatamoeba balamuthi, comprising together with them and aerobic lobose amoebae Vannella, Acanthamoeba, Balamuthia, and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens, does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of 'Archezoa': Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic 'crown' group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.     

Ultrastructure and ribosomal RNA phylogeny of the free-living heterotrophic flagellate Dysnectes brevis n. gen., n. sp., a new member of the Fornicata

Dysnectes brevis n. gen., n. sp., a free-living heterotrophic flagellate that grows under microaerophilic conditions possesses two flagella. The posterior one lies in a ventral feeding groove, suggesting that this flagellate is an excavate. Our detailed electron microscopic observations revealed that D. brevis possesses all the key ultrastructural characters considered typical of Excavata. Among the 10 excavate groups previously recognized, D. brevis displays an evolutionary affinity to members of the Fornicata (i.e. Carpediemonas, retortamonads, and diplomonads). Firstly, a strong D. brevis-Fornicata affinity was recovered in the phylogenetic analyses of small subunit ribosomal RNA (SSU rRNA) sequences, albeit the internal branching pattern of the D. brevis+Fornicata clade was not resolved with confidence. Corresponding to the SSU rRNA phylogeny, D. brevis and the Fornicata shared the following components of the flagellar apparatus: the arched B fiber bridging the right root; a posterior basal body; and a left root. Combining both morphological and molecular phylogenetic analyses, D. brevis is classified as a new free-living excavate in the Fornicata incertae sedis.     

Comprehensive multigene phylogenies of excavate protists reveal the evolutionary positions of "primitive" eukaryotes

Many of the protists thought to represent the deepest branches on the eukaryotic tree are assigned to a loose assemblage called the "excavates." This includes the mitochondrion-lacking diplomonads and parabasalids (e.g., Giardia and Trichomonas) and the jakobids (e.g., Reclinomonas). We report the first multigene phylogenetic analyses to include a comprehensive sampling of excavate groups (six nuclear-encoded protein-coding genes, nine of the 10 recognized excavate groups). Excavates coalesce into three clades with relatively strong maximum likelihood bootstrap support. Only the phylogenetic position of Malawimonas is uncertain. Diplomonads, parabasalids, and the free-living amitochondriate protist Carpediemonas are closely related to each other. Two other amitochondriate excavates, oxymonads and Trimastix, form the second monophyletic group. The third group is comprised of Euglenozoa (e.g., trypanosomes), Heterolobosea, and jakobids. Unexpectedly, jakobids appear to be specifically related to Heterolobosea. This tree topology calls into question the concept of Discicristata as a supergroup of eukaryotes united by discoidal mitochondrial cristae and makes it implausible that jakobids represent an independent early-diverging eukaryotic lineage. The close jakobids-Heterolobosea-Euglenozoa connection demands complex evolutionary scenarios to explain the transition between the presumed ancestral bacterial-type mitochondrial RNA polymerase found in jakobids and the phage-type protein in other eukaryotic lineages, including Euglenozoa and Heterolobosea.     

Primary Structure and Phylogenetic Relationships of Glyceraldehyde-3-Phosphate Dehydrogenase Genes of Free-Living and Parasitic Diplomonad Flagellates

ABSTRACT. Complete nucleotide sequences have been established for two genes (gap1 and gap2) coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) homologs in the diplomonad Giardia lamblia. In addition, almost complete sequences of the GAPDH open reading frames were obtained from PCR products for two free-living diplomonad species, Trepomonas agilis and Hexamita inflata, and a parasite of Atlantic salmon, an as yet unnamed species with morphological affinities to Spironucleus. Giardia lamblia gap 1 and the genes from the three other diplomonad species show high similarity to each other and to other glycolytic GAPDH genes. All amino-acyl residues known to be highly conserved in this enzyme are also conserved in these sequences. Giardia lamblia gap2 gene is more divergent and its putative translation reveals the presence of a cysteine and serine-rich insertion resembling a metal binding finger. This motif has not yet been noted in other GAPDH molecules. All sequences contain an S-loop signature with characteristics close to those of eukaryotes. In phylogenetic reconstructions based on the derived amino acid sequences with neighborjoining, parsimony and maximum-likelihood methods the four typical GAPDH sequences of diplomonads cluster into a single clade. Within this clade, G. lamblia gap1 shares a common ancestor with the rest of the genes. The latter are more closely related to each other, indicating an early separation of the lineage leading to the genus Giardia from the lineage encompassing the morphologically less differentiated genera, Trepomonas, Hexamita and that of the unnamed species. This result is discordant with the orthogonal evolution of diplomonads suggested on the basis of comparative morphology. In neighbor-joining reconstructions G. lamblia gap2 occupies a variable position, due to its great divergence. In parsimony and maximum likelihood analysis however, it shares a most recent common ancestor with the typical G. lamblia gap1 gene, suggesting that it diverged after the separation of the Giardia lineage. The position of the diplomonad clade in broader phylogenetic reconstructions is firmly within the typical cytosolic glycolytic representatives of GAPDH of eukaryotes.     

The phylogenetic position of the Prolecithophora (Rhabditophora, 'Platyhelminthes')

Complete 18S ribosomal DNA (rDNA) sequences and partial 28S rDNA sequences from a selection of rhabditophoran taxa were obtained and used in combination with literature data to determine the phylogenetic position of the Prolecithophora and of two families sometimes included in the Prolecithophora, the Urastomidae and the Genostomatidae. The results are largely compatible with earlier molecular studies when supported clades are considered, and adjusting for the denser taxonomic sampling of this study. The position of the Proseriata is not compatible with the taxon Seriata, which is rejected. The Rhabdocoela excluding the Fecampiida and the Neodermata is monophyletic. The phylogenetic position of the Neodermata cannot be determined, but its placement is not compatible with the proposed taxa Revertospermata and Mediofusata Kornakova & Joffe, 1999, which are rejected. The Urastomidae and the Genostomatidae in all analyses group with the Fecampiida, and it is our recommendation that these taxa be included in the Fecampiida. The amended Fecampiida always group separately from the Prolecithophora sensu stricto , the Rhabdocoela, and the Neodermata. Our analyses reveal the existence of a strongly supported clade consisting of Prolecithophora + Tricladida + the amended Fecampiida, and we propose the name Adiaphanida for this clade. Tentatively the sister group of the Prolecithophora is a clade consisting of the Tricladida + amended Fecampiida.     

Myriapod monophyly and relationships among myriapod classes based on nearly complete 28S and 18S rDNA sequences

Myriapods play a pivotal position in the arthropod phylogenetic tree. The monophyly of Myriapoda and its internal relationships have been difficult to resolve. This study combined nearly complete 28S and 18S ribosomal RNA gene sequences (3,826 nt in total) to estimate the phylogenetic position of Myriapoda and phylogenetic relationships among four myriapod classes. Our data set consists of six new myriapod sequences and homologous sequences for 18 additional species available in GenBank. Among the six new myriapod sequences, those of the one pauropod and two symphylans are very important additions because they were such difficult taxa to classify in past molecular-phylogenetic studies. Phylogenetic trees were constructed with maximum parsimony, maximum likelihood, and Bayesian analyses. All methods yielded moderate to strong support for the monophyly of Myriapoda. Symphyla grouped strongly with Pauropoda under all analytical conditions. The KH test rejected the traditional view of Dignatha and Progoneata, and the topology obtained here, though not significantly supported, was Diplopoda versus ((Symphyla + Pauropoda) + Chilopoda).     

Light Microscopic Observations, Ultrastructure, and Molecular Phylogeny of Hicanonectes teleskopos n. g., n. sp., a Deep-Branching Relative of Diplomonads

ABSTRACT. We describe Hicanonectes teleskopos n. g., n. sp., a heterotrophic flagellate isolated from low-oxygen marine sediment. Hicanonectes teleskopos has a ventral groove and two unequal flagella, and rapidly rotates during swimming. At the ultrastructural level H. teleskopos is a "typical excavate": it displays flagellar vanes, a split right microtubular root, "I,""B," and "C" fibres, a singlet microtubular root, and a possible composite fibre. Small subunit rRNA (SSU rRNA) gene phylogenies and an "arched" B fibre demonstrate that H. teleskopos belongs to Fornicata (i.e. diplomonads, retortamonads, and relatives). It forms a clade with the deep-branching fornicate Carpediemonas , with moderate-to-strong bootstrap support, although their SSU rRNA gene sequences are quite dissimilar. Hicanonectes differs from Carpediemonas in cell shape, swimming behaviour, number of basal bodies (i.e. 4 vs. 3), number of flagellar vanes (i.e. 2 vs. 3), anterior root organization, and by having a cytopharynx. Like Carpediemonas and Dysnectes, Hicanonectes has conspicuous mitochondrion-like organelles that lack cristae and superficially resemble the hydrogenosomes of parabasalids, rather than the mitosomes of their closer relatives the diplomonads (e.g. Giardia ).