Phylogenetic Analysis of Reptilian Hemoglobins: Trees, Rates, and Divergences

Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated α chains into α^A and α^D types, which are present in all reptiles except crocodiles, where only α^A chains are expressed. The occurrence of the α^D chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of β chains (βI and βII), while only one type of β chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both α and β sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon+ squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate α^A and α^D chains and crocodilian β chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (α^A) and 3.3 (β) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate ↔ sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved ∼3.5 (α^A) or ∼1.7 times (β) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times. Received: 15 September 1997 / Accepted: 4 February 1998     

Silica, Alumina, and Clay-Catalyzed Alanine Peptide Bond Formation

The peptide bond formation of alanine (ala), ala + glycine (gly), ala + diglycine (gly₂), and ala + gly cyclic anhydride (cyc-gly₂) in drying/wetting cycles at 80°C was studied. Silica, alumina, and representative smectites—montmorillonite and hectorite—were used as catalysts, and the dependence of reaction yields on the available amount of water in the reaction systems was evaluated. Silica and alumina catalyze the formation of oligopeptide mainly in temperature fluctuation experiments, whereas higher amounts of water in the reaction system support clay-catalyzed reactions. Silica and alumina are much more efficient for amino acid dimerization than clays. Whereas only 0.1% of ala oligomerized on hectorite and no reaction proceeded on montmorillonite, about 0.9 and 3.8% alanine converted into its dimer and cyclic anhydride on silica and alumina, respectively. Clay minerals, on the other hand, seem to more efficiently catalyze peptide chain elongation than amino acid dimerization. The reaction yields of ala-gly-gly and gly-gly-ala from ala + gly₂ and ala + cyc-gly₂ reached about 0.3% on montmorillonite and 1.0% on hectorite. The possible mechanisms of these reactions and the relevance of the results for prebiotic chemistry are discussed. Received: 15 December 1996 / Accepted: 1 May 1997     

ApA Dinucleotide Periodicity in Prokaryote, Eukaryote, and Organelle Genomes

Computer analyses of various genome sequences revealed the existence of certain periodical patterns of adenine–adenine dinucleotides (ApA). For each genome sequence of 13 eubacteria, 3 archaebacteria, 10 eukaryotes, 60 mitochondria, and 9 chloroplasts, we counted frequencies of ApA dinucleotides at each downstream position within 50 bp from every ApA. We found that the complete genomes of all three archaebacteria have clear ApA periodicities of about 10 bps. On the other hand, all of the 13 eubacteria we analyzed were found to have an ApA periodicity of about 11 bp. Similar periodicities exist in the 10 eukaryotes, although higher organisms such as primates tend to have weaker periodic patterns. None of the mitochondria and chroloplasts we analyzed showed an evident periodic pattern. Received: 3 November 1998 / Accepted: 24 March 1999     

Complete large subunit ribosomal RNA sequences from the heterokont algae ochromonas danica, nannochloropsis salina, and tribonema aequale, and phylogenetic analysis

The large subunit ribosomal RNA sequences from the heterokont algae Ochromonas danica, Nannochloropsis salina, and Tribonema aequale were determined. These sequences were combined with small subunit ribosomal RNA sequences in order to carry out a phylogenetic analysis based on neighbor-joining, maximum parsimony, and maximum likelihood methods. Our results indicate that heterokont fungi and heterokont algae each are monophyletic, and confirm that they together form a monophyletic group called ``stramenopiles.' Within the heterokont algae, the eustigmatophyte Nannochloropsis salina either clusters with the chrysophyte Ochromonas danica or forms a sister group to a cluster comprising the phaeophyte Scytosiphon lomentaria and the xanthophyte Tribonema aequale. The alveolates were identified as the closest relatives of the stramenopiles, but the exact order of divergence between the eukaryotic crown taxa could not be established with confidence. Received: 22 November 1996 / Accepted: 14 February 1997     

Obcells as Proto-Organisms: Membrane Heredity, Lithophosphorylation, and the Origins of the Genetic Code, the First Cells, and Photosynthesis

I attempt to sketch a unified picture of the origin of living organisms in their genetic, bioenergetic, and structural aspects. Only selection at a higher level than for individual selfish genes could power the cooperative macromolecular coevolution required for evolving the genetic code. The protein synthesis machinery is too complex to have evolved before membranes. Therefore a symbiosis of membranes, replicators, and catalysts probably mediated the origin of the code and the transition from a nucleic acid world of independent molecular replicators to a nucleic acid/protein/lipid world of reproducing organisms. Membranes initially functioned as supramolecular structures to which different replicators attached and were selected as a higher-level reproductive unit: the proto-organism. I discuss the roles of stereochemistry, gene divergence, codon capture, and selection in the code's origin. I argue that proteins were primarily structural not enzymatic and that the first biological membranes consisted of amphipathic peptidyl-tRNAs and prebiotic mixed lipids. The peptidyl-tRNAs functioned as genetically-specified lipid analogues with hydrophobic tails (ancestral signal peptides) and hydrophilic polynucleotide heads. Protoribosomes arose from two cooperating RNAs: peptidyl transferase (large subunit) and mRNA-binder (small subunit). Early proteins had a second key role: coupling energy flow to the phosphorylation of gene and peptide precursors, probably by lithophosphorylation by membrane-anchored kinases scavenging geothermal polyphosphate stocks. These key evolutionary steps probably occurred on the outer surface of an `inside out-cell' or obcell, which evolved an unambiguous hydrophobic code with four prebiotic amino acids and proline, and initiation by isoleucine anticodon CAU; early proteins and nucleozymes were all membrane-attached. To improve replication, translation, and lithophosphorylation, hydrophilic substrate-binding and catalytic domains were later added to signal peptides, yielding a ten-acid doublet code. A primitive proto-ecology of molecular scavenging, parasitism, and predation evolved among obcells. I propose a new theory for the origin of the first cell: fusion of two cup-shaped obcells, or hemicells, to make a protocell with double envelope, internal genome and ribosomes, protocytosol, and periplasm. Only then did water-soluble enzymes, amino acid biosynthesis, and intermediary metabolism evolve in a concentrated autocatalytic internal cytosolic soup, causing 12 new amino acid assignments, termination, and rapid freezing of the 22-acid code. Anticodons were recruited sequentially: GNN, CNN, INN, and *UNN. CO₂ fixation, photoreduction, and lipid synthesis probably evolved in the protocell before photophosphorylation. Signal recognition particles, chaperones, compartmented proteases, and peptidoglycan arose prior to the last common ancestor of life, a complex autotrophic, anaerobic green bacterium. Received: 19 February 2001 / Accepted: 9 April 2001     

Phylogenetic Relationships of Fungi, Plantae, and Animalia Inferred from Homologous Comparison of Ribosomal Proteins

The complete set of available ribosomal proteins was utilized, at both the peptidic and the nucleotidic level, to establish that plants and metazoans form two sister clades relative to fungi. Different phylogenetic inference methods are applied to the sequence data, using archeans as the outgroup. The evolutionary length of the internal branch within the eukaryotic crown trichotomy is demonstrated to be, at most, one-tenth of the evolutionary length of the branch leading to the cenancester of these three kingdoms. Received: 1 November 1997 / Accepted: 7 January 1998     

Phylogenetic relationships of annelids,molluscs, and arthropods evidenced from molecules and morphology

Annelids and arthropods have long been considered each other's closest relatives, as evidenced by similarities in their segmented body plans. An alternative view, more recently advocated by investigators who have examined partial 18S ribosomal RNA data, proposes that annelids, molluscs, and certain other minor phyla with trochophore larva stages share a more recent common ancestor with one another than any do with arthropods. The two hypotheses are mutually exclusive in explaining spiralian relationships. Cladistic analysis of morphological data does not reveal phylogentic relationships among major spiralian taxa but does suggest monophyly for both the annelids and molluscs. Distance and maximum-likelihood analyses of 18S rRNA gene sequences from major spiralian taxa suggest a sister relationship between annelids and molluscs and provide a clear resolution within the major groups of the spiralians. The parsimonious tree based on molecular data, however, indicates a sister relationship of the Annelida and Bivalvia, and an earlier divergence of the Gastropoda than the Annelida–Bivalvia clade. To test further hypotheses on the phylogenetic relationships among annelids, molluscs, and arthropods, and the ingroup relationships within the major spiralian taxa, we combine the molecular and morphological data sets and subject the combined data matrix to parsimony analysis. The resulting tree suggests that the molluscs and annelids form a monophyletic lineage and unites the molluscan taxa to a monophyletic group. Therefore, the result supports the Eutrochozoa hypothesis and the monophyly of molluscs, and indicates early acquisition of segmented body plans in arthropods. Received: 25 September 1995 / Accepted: 15 March 1996     

Apolipophorin II/I, Apolipoprotein B, Vitellogenin, and Microsomal Triglyceride Transfer Protein Genes Are Derived from a Common Ancestor

Large lipid transfer proteins (LLTP) are nonexchangeable apolipoproteins and intracellular lipid-exchange proteins involved in the assembly, secretion, and metabolism of lipoproteins. We have identified contiguous conserved sequence motifs in alignments of insect apolipophorin II/I precursor (apoLp-II/I), human apolipoprotein B (apoB), invertebrate and vertebrate vitellogenins (VTG), and the large subunit of mammalian microsomal triglyceride transfer protein (MTP). Conserved motifs present in the N-terminal part of nonexchangeable apolipoproteins encompass almost completely the large subunit of MTP, suggesting a derivation from a common ancestral functional unit, termed large lipid transfer (LLT) module. Divergence of LLTP from a common ancestor is supported by (1) the statistical significance of the combined match scores obtained after motif-based database searches, (2) the presence of several identical amino acid residues in all LLTP sequences currently available, (3) the conservation of hydrophobic clusters in an α-helical domain, (4) the phylogenetic analysis of the conserved sequences related to the von Willebrand factor D (VWD) module identified in nonexchangeable apolipoproteins, and (5) the presence of four and one ancestral exon boundaries in the LLT and VWD modules, respectively. Our data indicate that the genes coding for apoLp-II/I, apoB, VTG, and the MTP large subunit are members of the same multigene superfamily. LLTP have emerged from an ancestral molecule designed to ensure a pivotal event in the intracellular and extracellular transfer of lipids and liposoluble substances. Received: 8 June 1998 / Accepted: 15 February 1999     

Heat Shock Protein 70 Family: Multiple Sequence Comparisons, Function, and Evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus ``matches' 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins. Received: 29 January 1998 / Accepted: 14 May 1998     

Protein Tyrosine Kinase cDNAs from Amphioxus, Hagfish, and Lamprey: Isoform Duplications Around the Divergence of Cyclostomes and Gnathostomes

Animals evolved a variety of gene families involved in cell–cell communication and developmental control by gene duplication and domain shuffling. Each family is made up of several subtypes or subfamilies with distinct structures and functions, which diverged by gene duplications and domain shufflings before the divergence of parazoans and eumetazoans. Since the separation from protostomes, vertebrates expanded the multiplicity of members (isoforms) in the same subfamily by further gene duplications in their early evolution before the fish–tetrapod split. To know the dates of isoform duplications more closely, we have conducted isolation and sequencing cDNAs encoding the fibroblast growth factor receptor, Eph, src, and platelet-derived growth factor receptor subtypes belonging to the protein tyrosine kinase family from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Lampetra reissneri, a lamprey. From a phylogenetic tree of each subfamily inferred from a maximum likelihood (ML) method, together with a bootstrap analysis based on the ML method, we have shown that the isoform duplications frequently occurred in the early evolution of vertebrates around or just before the divergence of cyclostomes and gnathostomes by gene duplications and possibly chromosomal duplications. Received: 28 April 1998 / Accepted: 30 June 1999