Actin and ubiquitin protein sequences support a cercozoan/foraminiferan ancestry for the plasmodiophorid plant pathogens

The plasmodiophorids are a group of eukaryotic intracellular parasites that cause disease in a variety of economically significant crops. Plasmodiophorids have traditionally been considered fungi but have more recently been suggested to be members of the Cercozoa, a morphologically diverse group of amoeboid, flagellate, and amoeboflagellate protists. The recognition that Cercozoa constitute a monophyletic lineage has come from phylogenetic analyses of small subunit ribosomal RNA genes. Protein sequence data have suggested that the closest relatives of Cercozoa are the Foraminifera. To further test a cercozoan origin for the plasmodiophorids, we isolated actin genes from Plasmodiophora brassicae, Sorosphaera veronicae, and Spongospora subterranea, and polyubiquitin gene fragments from P. brassicae and S. subterranea. We also isolated actin genes from the chlorarachniophyte Lotharella globosa. In protein phylogenies of actin, the plasmodiophorid sequences consistently branch with Cercozoa and Foraminifera, and weakly branch as the sister group to the foraminiferans. The plasmodiophorid polyubiquitin sequences contain a single amino acid residue insertion at the functionally important processing point between ubiquitin monomers, the same place in which an otherwise unique insertion exists in the cercozoan and foraminiferan proteins. Taken together, these results indicate that plasmodiophorids are indeed related to Cercozoa and Foraminifera, although the relationships amongst these groups remain unresolved.     

A novel polyubiquitin structure in Cercozoa and Foraminifera: evidence for a new eukaryotic supergroup

Ubiquitin is a 76 amino acid protein with a remarkable degree of evolutionary conservation. Ubiquitin plays an essential role in a large number of eukaryotic cellular processes by targeting proteins for proteasome-mediated degradation. Most ubiquitin genes are found as head-to-tail polymers whose products are posttranslationally processed to ubiquitin monomers. We have characterized polyubuiquitin genes from the photosynthetic amoeboflagellate Chlorarachnion sp. CCMP 621 (also known as Bigelowiella natans) and found that they deviate from the canonical polyubiquitin structure in having an amino acid insertion at the junction between each monomer, suggesting that polyubiquitin processing in this organism is unique among eukaryotes. The gene structure indicates that processing likely cleaves monomers at the amino terminus of the insertion. We examined the phylogenetic distribution of the insertion by sequencing polyubiquitin genes from several other eukaryotic groups and found it to be confined to Cercozoa (including Chlorarachnion, Lotharella, Cercomonas, and Euglypha) and Foraminifera (including Reticulomyxa and Haynesina). This character strongly suggests that Cercozoa and Foraminifera are close relatives and form a new "supergroup" of eukaryotes.     

Revised small subunit rRNA analysis provides further evidence that Foraminifera are related to Cercozoa

There is accumulating evidence that the general shape of the ribosomal DNA-based phylogeny of Eukaryotes is strongly biased by the long-branch attraction phenomenon, leading to an artifactual basal clustering of groups that are probably highly derived. Among these groups, Foraminifera are of particular interest, because their deep phylogenetic position in ribosomal trees contrasts with their Cambrian appearance in the fossil record. A recent actin-based phylogeny of Eukaryotes has proposed that Foraminifera might be closely related to Cercozoa and, thus, branch among the so-called crown of Eukaryotes. Here, we reanalyze the small-subunit ribosomal RNA gene (SSU rDNA) phylogeny by removing all long-branching lineages that could artifactually attract foraminiferan sequences to the base of the tree. Our analyses reveal that Foraminifera branch together with the marine testate filosean Gromia oviformis as a sister group to Cercozoa, in agreement with actin phylogeny. Our study confirms the utility of SSU rDNA as a phylogenetic marker of megaevolutionary history, provided that the artifacts due to the heterogeneity of substitution rates in ribosomal genes are circumvented.     

Foraminifera and Cercozoa are related in actin phylogeny: two orphans find a home?

In recent years, the increased sampling of protein-coding genes from diverse eukaryotes has revealed that many aspects of each gene tree are at odds with other phylogenies. This has led to the belief that each gene tree has unique strengths and weaknesses, suggesting that an accurate picture of eukaryotic relationships will be achieved only through comparative phylogeny using several different genes. To this end, actin genes were characterized from two genera of chlorarachniophytes, Chlorarachnion and Lotharella, and three species of the cercomonad flagellate Cercomonas: Phylogenetic trees including these new actin genes confirm the recently proposed relationship between chlorarachniophytes and cercomonads (Cercozoa) and, more importantly, also show a close relationship between Cercozoa and Foraminifera. Both of these are major eukaryotic groups encompassing extremely diverse organisms, yet there is no strong evidence for the evolutionary position of either from morphological or molecular data. The union of Cercozoa and Foraminifera suggested by actin phylogeny represents a novel step in the long process of determining the broad relationships between all major eukaryotic groups.     

Novel cultured protists identify deep-branching environmental DNA clades of cercozoa: New Genera Tremula, Micrometopion, Minimassisteria, Nudifila, Peregrinia

We describe three new orders of filosan Cercozoa, five new deep-branching genera, eight new species of Thaumatomonas, Reckertia, Spongomonas, Rhogostoma, Agitata, Neoheteromita and Paracercomonas, sequence their 18S rDNA, and construct 18S rDNA trees for 148 Cercozoa. Our phylogeny indicates that Filosa were ancestrally gliding flagellates; non-flagellate filose amoebae evolved from them five times independently. The new genera are more closely related to environmental DNA sequences than cultured organisms. Tremula longifila, a zooflagellate glider on both flagella (unlike other Cercozoa), is the most divergent filosan (Tremulida ord. n.). Micrometopion nutans is a eukaryote-eating gliding zooflagellate like Metopion and Metromonas. Minimassisteria diva is a widespread trimorphic marine amoeboflagellate granofilosan. Peregrinia clavideferens, a non-testate, scale-bearing, filose amoeba, branches deeply in Thaumatomonadida, which are probably sisters to Spongomonadida. Nudifila producta is a filose amoeboflagellate related to Clautriavia and Marimonadida (ord. n., e.g. Pseudopirsonia, Auranticordis). We substantially revise Imbricatea, now including Spongomonadida, and Thecofilosea to include Phaeodaria. Thecofilosea and Imbricatea and Thecofilosea are sisters, both arguably ancestrally rigid gliding flagellates with ventral pseudopod-emitting grooves. Scale-free Ovulinata parva is sister to Paulinella, so imbricate silica scales can be lost. Internal hollow silica skeletons evolved twice in Thecofilosea (Ebriida, Phaeodaria) or were multiply lost. Protaspa replaces preoccupied 'Protaspis'.     

Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA

Gromia oviformis Dujardin is a common marine protist characterised by a large, globular test and filose pseudopodia. First considered a foraminifer, Gromia was later placed within the Filosea and recently included among amoebae of uncertain affinities. In order to clarify the phylogenetic position of this genus, we sequenced the complete small-subunit ribosomal DNA gene of G. oviformis collected at five different geographic localities. The high divergence of obtained sequences suggests that G. oviformis is a species complex composed of several genetically distinct sibling species. Sequence analyses show Gromia to be a member of the Cercozoa, a heterogeneous assemblage which includes filose amoebae, the amoeboflagellate cercomonads, the chlorarachniophytes and the plasmodiophorid plant pathogens. Contrary to traditional classification, Gromia is not closely related to other testate filose amoebae (the Euglyphida), but seems to branch early among the Cercozoa. Our analyses also show a close relationship between the Cercozoa and the Acantharea. Because the Cercozoa are related to the Foraminifera based on other molecular data, we propose that most protists possessing filopodia, reticulopodia and axopodia have a common origin.     

Aurigamonas solis n. gen., n. sp., a soil-dwelling predator with unusual helioflagellate organisation and belonging to a novel clade within the Cercozoa

A flagellate predator, Aurigamonas solis n. gen., n. sp., with numerous radiating axopodia-like appendages, has been isolated in culture from soils. Despite its heliozoan-like appearance, Aurigamonas is not a sit-and-wait predator but a mobile hunter and its stiff appendages are not microtubule-supported axopodia but elongate haptopodia, each supported by a cylindrical core of microfilaments and bearing at its capitate tip a single extrusome-like body (haptosome). Prey flagellates are trapped on the sticky tips of the haptopodia and a large funnel-shaped pseudopodium then emerges to engulf the prey or suck out part of it for internal digestion. Pseudopodial contact is accompanied by killing, possibly as a result of the injection of spicules by the predator. Cytoplasmic haptosomes appear to induce formation of a haptopodium on making contact with the plasma membrane. Propulsion of the organism along the substratum is effected by beating of a long trailing flagellum, the short and inconspicuous second flagellum lacks motility. Small subunit rDNA sequencing shows that Aurigamonas arose within the Cercozoa. Its closest relatives are Cercobodo agilis and several flagellates currently known only as environmental sequences. This conclusion is supported further by the presence of only a single amino acid insertion in the polyubiquitin sequence of Aurigamonas solis.     

Phylogeny and classification of phylum Cercozoa (Protozoa)

The protozoan phylum Cercozoa embraces numerous ancestrally biciliate zooflagellates, euglyphid and other filose testate amoebae, chlorarachnean algae, phytomyxean plant parasites (e.g. Plasmodiophora, Phagomyxa), the animal-parasitic Ascetosporea, and Gromia. We report 18S rRNA sequences of 27 culturable zooflagellates, many previously of unknown taxonomic position. Phylogenetic analysis shows that all belong to Cercozoa. We revise cercozoan classification in the light of our analysis and ultrastructure, adopting two subphyla: Filosa subphyl. nov. a clade comprising Monadofilosa and Reticulofilosa, ranked as superclasses, ancestrally having the same very rare base-pair substitution as all opisthokonts; and subphylum Endomyxa emend. comprising classes Phytomyxea (Plasmodiophorida, Phagomyxida), Ascetosporea (Haplosporidia, Paramyxida, Claustrosporida ord. nov.) and Gromiidea cl. nov., which did not. Monadofilosa comprise Sarcomonadea, zooflagellates with a propensity to glide on their posterior cilium and/or generate filopodia (e.g. Metopion;Cercomonas; Heteromitidae – Heteromita, Bodomorpha, Proleptomonas and Allantion) and two new classes: Imbricatea (with silica scales: Euglyphida; Thaumatomonadida, including Allas, Thaumatomastix) and Thecofilosea (Cryomonadida; Tectofilosida ord. nov. – non-scaly filose amoebae, e.g. Pseudodifflugia). Reticulofilosa comprise classes Chlorarachnea, Spongomonadea and Proteomyxidea (e.g. Massisteria, Gymnophrys, a Dimorpha-like protozoan). Cercozoa, now with nine classes and 17 orders (four new), will probably include many, possibly most, other filose and reticulose amoebae and zooflagellates not yet assigned to phyla.     

Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata

Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of this huge eukaryotic group. Multiple protein sequences will be needed fully to resolve basal bikont phylogeny. Nonetheless, our results suggest that combined SSU+LSU rDNA phylogenies can help to resolve several ambiguous regions of the eukaryotic tree and identify key taxa for subsequent multi-gene analyses.     

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

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

Radiolaria divided into Polycystina and Spasmaria in combined 18S and 28S rDNA phylogeny

Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.     

Gene arrangements and branching orders of gram-positive bacteria

The availability of complete genomic sequence data allows one to develop new methods of reconstructing phylogenetic trees. A simple method of reconstructing branching orders based on gene transposition (or lateral transfer) is presented. It is argued that specific gene arrangements on four different genomes could determine a branching order. A computer search for such gene arrangements was carried out against gene order data of completely sequenced Gram-positive bacteria. Gene arrangements around ribosomal protein S4 gene, murC (UDP-N-acetylmuramate:alanine ligase) gene and dnaE (DNA polymerase III alpha chain) gene each suggest a branching order in which actinobacteria with a high genomic G+C content first branched off from other Gram-positives with a low G+C content and then a split occurred between Mycoplasma species and a group closely related to Bacillus subtilis. A recently sequenced thermophilic bacterium Thermoanaerobacter tengcongensis is suggested to have branched off from the lineage leading to the low G+C Gram-positives prior to the split between the Mycoplasma and Bacillus groups. By contrast to the indel analysis in which a single evolutionary event of insertion or deletion of a signature sequence is assumed, the present method does not necessarily require such a parsimonious assumption of gene transposition.     

Phylogenetic Analysis of Eukaryotes Using Heat-Shock Protein Hsp90

Most eukaryote molecular phylogenies have been based on small-subunit ribosomal RNA as its database includes the most species, but serious problems have been encountered that can make these trees misleading. More recent studies using concatenated protein sequences have increased the data per organism, reducing misleading signals from a single sequence, but taxon sampling is limited. To increase the database of protein-coding genes we sequenced the cytosolic form of heat-shock protein Hsp90 from a broad variety of previously unsampled eukaryote groups: protozoan flagellates (phyla Choanozoa, Apusozoa, Cercozoa) and all three groups of chromists (Cryptophyta, Heterokonta, Haptophyta). Gamma-corrected distance trees robustly show three groups: bacterial sequences are sister to all eukaryote sequences, which are cleanly subdivided into the cytosolic sequences and a clade comprising the chloroplast and endoplasmic reticulum (ER) Hsp90 sequences. The eukaryote cytosolic sequences comprise a robust opisthokont clade (animals/Choanozoa/fungi), a bikont clade, and an amoebozoan branch. However their topology is not robust. When the cytosolic sequences are rooted using only the ER/chloroplast clade as outgroup the amoebozoan Dictyostelium is sister to the opisthokonts forming a unikont clade in the distance tree. Congruence of this tree with that for concatenated mitochondrial proteins suggests that the root of the eukaryote tree is between unikonts and bikonts. Gamma-corrected maximum likelihood analyses of cytosolic sequences alone (519 unambiguously aligned amino acid positions) show bikonts as a clade, as do least-squares distance trees, but with other distance methods and parsimony the sole amoebozoan species branches weakly within bikonts. Choanozoa are clearly sisters to animals. Some major bikont groups (e.g. green plants, alveolates, Euglenozoa) are consistently recovered, but others (e.g. discicristates, chromalveolates) appear only in some trees; the backbone of the bikont subtree is not resolved, the position of groups represented only by single sequences being particularly unclear. Although single-gene trees will probably never resolve these uncertainties, the congruence of Hsp90 trees with other data is greater than for most other molecules and further taxon sampling of this molecule is recommended.     

Nucleotide sequence of the gene coding for the elongation factor Tu from the extremely thermophilic eubacterium Thermotoga maritima

Abstract The gene coding for the elongation factor Tu (EF-Tu) of Thermatoga maritima was cloned and sequenced. The predicted amino acid sequence was compared with those of other eubacteria, an archaebacterium and two eukaryotes as well. The similarity values and the distance matrix tree show that Thermotoga is more closely related to the eubacteria than to the representatives of the other urkingdoms. Thermotoga maritima represents the deepest branching within the tree of EF-Tu sequences from all eubacteria studied so far.     

Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes

The human and rat genes for a fifth muscarinic receptor have been cloned and expressed in mammalian cells. The 532 amino acid human protein has 89% sequence identity to the 531 amino acid rat protein and is most closely related to the m3 receptor. Both proteins are encoded by single exons. The receptor has intermediate affinity for pirenzepine and low affinity for AF-DX 116, and it increases metabolism of phosphatidylinositol when stimulated with carbachol. Expression of mRNA has yet to be observed in brain or selected peripheral tissues, suggesting that either it is substantially less abundant than m1-m4 or its distribution is quite different.     

Adaptive evolution of fertilization proteins within a genus: variation in ZP2 and ZP3 in deer mice (Peromyscus)

Rapid evolution of reproductive proteins has been documented in a wide variety of taxa. In internally fertilized species, knowledge about the evolutionary dynamics of these proteins between closely related taxa is primarily limited to accessory gland proteins in the semen of Drosophila. Investigation of additional taxa and functional classes of proteins is necessary in order to determine if there is a general pattern of adaptive evolution of reproductive proteins between recently diverged species. We performed an evolutionary analysis of 2 egg coat proteins, ZP2 and ZP3, in 15 species of deer mice (genus Peromyscus). Both of these proteins are involved in egg-sperm binding, a critical step in maintaining species-specific fertilization. Here, we show that Zp2 and Zp3 gene trees are not consistent with trees based on nonreproductive genes, Mc1r and Lcat, where species formed monophyletic clades. In fact, for both of the reproductive genes, intraspecific amino acid variation was extensive and alleles were sometimes shared across species. We document positive selection acting on ZP2 and ZP3 and identify specific amino acid sites that are likely targets of selection using both maximum likelihood approaches and patterns of parallel amino acid change. In ZP3, positively selected sites are clustered in and around the region implicated in sperm binding in Mus, suggesting changes may impact egg-sperm binding and fertilization potential. Finally, we identify lineages with significantly elevated rates of amino acid substitution using a Bayesian mapping approach. These findings demonstrate that the pattern of adaptive reproductive protein evolution found at higher taxonomic levels can be documented between closely related mammalian species, where reproductive isolation has evolved recently.     

Structural and energetic differences between insertions and substitutions in staphylococcal nuclease.

In a previous study, the small protein staphylococcal nuclease was shown to readily accommodate single alanine and glycine insertions, with average losses in stability comparable to substitutions at the same sites (PROT. 7:299-305, 1990). To more fully explore this unexpected adaptability to changes in residue spacing, 2 double amino acid insertions (alanyl-glycine, glycyl-glycine) and 3 additional single amino acid insertions with dissimilar side chains (proline, leucine, and glutamine) were constructed at 10 of the sites previously studied. At 8 of these sites, the type of amino acid side chain on the inserted residue significantly influenced the stability of the mutant protein. However, at 9 of the 10 sites, the double insertions were found to be no more destabilizing than the single alanine or glycine insertions. In contrast, double substitution mutations of staphylococcal nuclease, which replace two adjacent residues with alanine, do not show this striking degree of non-additivity. A comparison of the effects of single glutamine and single glycine insertions with alanyl-glycine insertions indicates that insertion of alanine into the peptide backbone is, on average, less destabilizing than appending the equivalent atoms onto the side chain of a glycine insertion. To explain their very different energetic effects, we propose that, unlike most substitutions, the inserted residue(s) must induce lateral displacements of the polypeptide chain, forcing the folded conformation away from that of wild type. The resulting obligatory shifts in the positioning of residues flanking the insertion generate a large number of degrees of freedom around which the mutant structure can relax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phylogenetic extinction rates and comparative methodology

Species are not independent points for comparative analyses because closely related species share more evolutionary history and are therefore more similar to each other than distantly related species. The extent to which independent-contrast analysis reduces type I and type II statistical error in comparison with cross-species analysis depends on the relative branch lengths in the phylogenetic tree: as deeper branches get relatively long, cross-species analyses have more statistical type I and type II error. Phylogenetic trees reconstructed from extant species, under the assumptions of a branching process with speciation (branching) and extinction rates remaining constant through time, will have relatively longer deep branches as the extinction rate increases relative to the speciation rate. We compare the statistical performance of cross-species and independent-contrast analyses with varying relative extinction rates, and conclude that cross-species comparisons have unacceptable statistical performance, particularly when extinction rates are relatively high.     

Conflicting phylogenetic signals at the base of the metazoan tree

A phylogenetic framework is essential for under-standing the origin and evolution of metazoan development. Despite a number of recent molecular studies and a rich fossil record of sponges and cnidarians, the evolutionary relationships of the early branching metazoan groups to each other and to a putative outgroup, the choanoflagellates, remain uncertain. This situation may be the result of the limited amount of phylogenetic information found in single genes and the small number of relevant taxa surveyed. To alleviate the effect of these analytical factors in the phylogenetic recons-truction of early branching metazoan lineages, we cloned multiple protein-coding genes from two choanoflagellates and diverse sponges, cnidarians, and a ctenophore. Comparisons of sequences for alpha-tubulin, beta-tubulin, elongation factor 2, HSP90, and HSP70 robustly support the hypothesis that choanoflagellates are closely affiliated with animals. However, analyses of single and concatenated amino acid sequences fail to resolve the relationships either between early branching metazoan groups or between Metazoa and choano-flagellates. We demonstrate that variable rates of evolution among lineages, sensitivity of the analyses to taxon selection, and conflicts in the phylogenetic signal contained in different amino acid sequences obscure the phylogenetic associations among the early branching Metazoa. These factors raise concerns about the ability to resolve the phylogenetic history of animals with molecular sequences. A consensus view of animal evolution may require investigations of genome-scale characters.