Multiple independent origins of mitochondrial gene order in birds

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced OL (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.

Molecular evidence for an African origin of the Hawaiian endemic Hesperomannia (Asteraceae)

Identification of the progenitors of plants endemic to oceanic islands often is complicated by extreme morphological divergence between island and continental taxa. This is especially true for the Hawaiian Islands, which are 3,900 km from any continental source. We examine the origin of Hesperomannia, a genus of three species endemic to Hawaii that always have been placed in the tribe Mutisieae of the sunflower family. Phylogenetic analyses of representatives from all tribes in this family using the chloroplast gene ndhF (where ndhF is the ND5 protein of chloroplast NADH dehydrogenase) indicate that Hesperomannia belongs to the tribe Vernonieae. Phylogenetic comparisons within the Vernonieae using sequences of both ndhF and the internal transcribed spacer regions of nuclear ribosomal DNA reveal that Hesperomannia is sister to African species of Vernonia. Long-distance dispersal northeastward from Africa to southeast Asia and across the many Pacific Ocean island chains is the most likely explanation for this unusual biogeographic connection. The 17- to 26-million-year divergence time between African Vernonia and Hesperomannia estimated by the DNA sequences predates the age of the eight existing Hawaiian Islands. These estimates are consistent with an hypothesis that the progenitor of Hesperomannia arrived at one of the low islands of the Hawaiian-Emperor chain between the late Oligocene and mid-Miocene when these islands were above sea level. Subsequent to its arrival the southeast Pacific island chains served as steppingstones for dispersal to the existing Hawaiian Islands.

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix–loop–helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

LGICdb: the ligand-gated ion channel database

Ligand-Gated Ion Channels (LGIC) are polymeric transmembrane proteins involved in the fast response to numerous neurotransmitters. All these receptors are formed by homologous subunits and the last two decades revealed an unexpected wealth of genes coding for these subunits. The Ligand-Gated Ion Channel database (LGICdb) has been developed to handle this increasing amount of data. The database aims to provide only one entry for each gene, containing annotated nucleic acid and protein sequences. The repository is carefully structured and the entries can be retrieved by various criteria. In addition to the sequences, the LGICdb provides multiple sequence alignments, phylogenetic analyses and atomic coordinates when available. The database is accessible via the World Wide Web (http://www.pasteur.fr/recherche/banques/LGIC/LGIC.html), where it is continuously updated. The version 16 (September 2000) available for download contained 333 entries covering 34 species.

Feeding specialization and host-derived chemical defense in Chrysomeline leaf beetles did not lead to an evolutionary dead end

Combination of molecular phylogenetic analyses of Chrysomelina beetles and chemical data of their defensive secretions indicate that two lineages independently developed, from an ancestral autogenous metabolism, an energetically efficient strategy that made the insect tightly dependent on the chemistry of the host plant. However, a lineage (the interrupta group) escaped this subordination through the development of a yet more derived mixed metabolism potentially compatible with a large number of new host-plant associations. Hence, these analyses on leaf beetles document a mechanism that can explain why high levels of specialization do not necessarily lead to “evolutionary dead ends.”

Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions

The evolution of novelty in tightly integrated biological systems, such as hormones and their receptors, seems to challenge the theory of natural selection: it has not been clear how a new function for any one part (such as a ligand) can be selected for unless the other members of the system (e.g., a receptor) are already present. Here I show—based on identification and phylogenetic analysis of steroid receptors in basal vertebrates and reconstruction of the sequences and functional attributes of ancestral proteins—that the first steroid receptor was an estrogen receptor, followed by a progesterone receptor. Genome mapping and phylogenetic analyses indicate that the full complement of mammalian steroid receptors evolved from these ancient receptors by two large-scale genome expansions, one before the advent of jawed vertebrates and one after. Specific regulation of physiological processes by androgens and corticoids are relatively recent innovations that emerged after these duplications. These findings support a model of ligand exploitation in which the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances. In this way, novel hormone-receptor pairs are created, and an integrated system of increasing complexity elaborated. This model suggests that ligands for some “orphan” receptors may be found among intermediates in the synthesis of ligands for phylogenetically related receptors.

Evolution by the birth-and-death process in multigene families of the vertebrate immune system

Concerted evolution is often invoked to explain the diversity and evolution of the multigene families of major histocompatibility complex (MHC) genes and immunoglobulin (Ig) genes. However, this hypothesis has been controversial because the member genes of these families from the same species are not necessarily more closely related to one another than to the genes from different species. To resolve this controversy, we conducted phylogenetic analyses of several multigene families of the MHC and Ig systems. The results show that the evolutionary pattern of these families is quite different from that of concerted evolution but is in agreement with the birth-and-death model of evolution in which new genes are created by repeated gene duplication and some duplicate genes are maintained in the genome for a long time but others are deleted or become nonfunctional by deleterious mutations. We found little evidence that interlocus gene conversion plays an important role in the evolution of MHC and Ig multigene families.

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Two highly homologous ribonuclease genes expressed in mouse eosinophils identify a larger subgroup of the mammalian ribonuclease superfamily.

Two putative ribonucleases have been isolated from the secondary granules of mouse eosinophils. Degenerate oligonucleotide primers inferred from peptide sequence data were used in reverse transcriptase-PCR reactions of bone marrow-derived cDNA. The resulting PCR product was used to screen a C57BL/6J bone marrow cDNA library, and comparisons of representative clones showed that these genes and encoded proteins are highly homologous (96% identity at the nucleotide level; 92/94% identical/similar at the amino acid level). The mouse proteins are only weakly homologous (approximately 50% amino acid identity) with the human eosinophil-associated ribonucleases (i.e., eosinophil-derived neurotoxin and eosinophil cationic protein) and show no sequence bias toward either human protein. Phylogenetic analyses established that the human and mouse loci shared an ancestral gene, but that independent duplication events have occurred since the divergence of primates and rodents. The duplication event generating the mouse genes was estimated to have occurred < 5 x 10(6) years ago (versus 30 to 40 x 10(6) years ago in primates). The identification of independent duplication events in two extant mammalian orders suggests a selective advantage to having multiple eosinophil granule ribonucleases. Southern blot analyses in the mouse demonstrated the existence of three additional highly homologous genes (i.e., five genes total) as well as several more divergent family members. The potential significance of this observation is the implication of a larger gene subfamily in primates (i.e., humans).

A novel clade of protistan parasites near the animal-fungal divergence.

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.

The evolution of the vertebrate Dlx gene family.

The vertebrate Dlx gene family consists of homeobox-containing transcription factors distributed in pairs on the same chromosomes as the Hox genes. To investigate the evolutionary history of Dlx genes, we have cloned five new zebrafish family members and have provided additional sequence information for two mouse genes. Phylogenetic analyses of Dlx gene sequences considered in the context of their chromosomal arrangements suggest that an initial tandem duplication produced a linked pair of Dlx genes after the divergence of chordates and arthropods but prior to the divergence of tunicates and vertebrates. This pair of Dlx genes was then duplicated in the chromosomal events that led to the four clusters of Hox genes characteristic of bony fish and tetrapods. It is possible that a pair of Dlx genes linked to the Hoxc cluster has been lost from mammals. We were unable to distinguish between independent duplication and retention of the ancestral state of bony vertebrates to explain the presence of a greater number of Dlx genes in zebrafish than mammals. Determination of the linkage relationship of these additional zebrafish Dlx genes to Hox clusters should help resolve this issue.

A common evolutionary origin for mitochondria and hydrogenosomes.

Trichomonads are among the earliest eukaryotes to diverge from the main line of eukaryotic descent. Keeping with their ancient nature, these facultative anaerobic protists lack two "hallmark" organelles found in most eukaryotes: mitochondria and peroxisomes. Trichomonads do, however, contain an unusual organelle involved in carbohydrate metabolism called the hydrogenosome. Like mitochondria, hydrogenosomes are double-membrane bounded organelles that produce ATP using pyruvate as the primary substrate. Hydrogenosomes are, however, markedly different from mitochondria as they lack DNA, cytochromes and the citric acid cycle. Instead, they contain enzymes typically found in anaerobic bacteria and are capable of producing molecular hydrogen. We show here that hydrogenosomes contain heat shock proteins, Hsp70, Hsp60, and Hsp10, with signature sequences that are conserved only in mitochondrial and alpha-Gram-negative purple bacterial Hsps. Biochemical analysis of hydrogenosomal Hsp60 shows that the mature protein isolated from the organelle lacks a short, N-terminal sequence, similar to that observed for most nuclear-encoded mitochondrial matrix proteins. Moreover, phylogenetic analyses of hydrogenosomal Hsp70, Hsp60, and Hsp10 show that these proteins branch within a monophyletic group composed exclusively of mitochondrial homologues. These data establish that mitochondria and hydrogenosomes have a common eubacterial ancestor and imply that the earliest-branching eukaryotes contained the endosymbiont that gave rise to mitochondria in higher eukaryotes.

Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor.

The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.

Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.

Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA.

An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.