Duplication and divergence of the genes of the α-esterase cluster ofDrosophila melanogaster

The α-esterase cluster of D. melanogaster contains 11 esterase genes dispersed over 60 kb. Embedded in the cluster are two unrelated open reading frames that have sequence similarity with genes encoding ubiquitin-conjugating enzyme and tropomyosin. The esterase amino acid sequences show 37–66% identity with one another and all but one have all the motifs characteristic of functional members of the carboxyl/cholinesterase multigene family. The exception has several frameshift mutations and appears to be a pseudogene. Patterns of amino acid differences among cluster members in relation to generic models of carboxyl/cholinesterase protein structure are broadly similar to those among other carboxyl/cholinesterases sequenced to date. However the α-esterases differ from most other members of the family in: their lack of a signal peptide; the lack of conservation in cysteines involved in disulfide bridges; and in four indels, two of which occur in or adjacent to regions that align with proposed substrate-binding sites of other carboxyl/cholinesterases. Phylogenetic analyses clearly identify three simple gene duplication events within the cluster. The most recent event involved the pseudogene which is located in an intron of another esterase gene. However, relative rate tests suggest that the pseudogene remained functional after the duplication event and has become inactive relatively recently. The distribution of indels also suggests a deeper node in the gene phylogeny that separates six genes at the two ends of the cluster from a block of five in the middle. Received: 18 January 1996 / Accepted: 12 March 1996     

Molecular Evolution of Nitrogen Fixation: The Evolutionary History of the nifD, nifK, nifE, and nifN Genes

The pairs of nitrogen fixation genes nifDK and nifEN encode for the α and β subunits of nitrogenase and for the two subunits of the NifNE protein complex, involved in the biosynthesis of the FeMo cofactor, respectively. Comparative analysis of the amino acid sequences of the four NifD, NifK, NifE, and NifN in several archaeal and bacterial diazotrophs showed extensive sequence similarity between them, suggesting that their encoding genes constitute a novel paralogous gene family. We propose a two-step model to reconstruct the possible evolutionary history of the four genes. Accordingly, an ancestor gene gave rise, by an in-tandem paralogous duplication event followed by divergence, to an ancestral bicistronic operon; the latter, in turn, underwent a paralogous operon duplication event followed by evolutionary divergence leading to the ancestors of the present-day nifDK and nifEN operons. Both these paralogous duplication events very likely predated the appearance of the last universal common ancestor. The possible role of the ancestral gene and operon in nitrogen fixation is also discussed. Received: 21 June 1999 / Accepted: 1 March 2000     

Characterization of Two Alcohol Dehydrogenase (Adh) Loci from the Olive Fruit Fly, Bactrocera (Dacus) oleae and Implications for Adh Duplication in Dipteran Insects

We report the cloning and structural characterization of two Adh loci of the olive fruit fly, Bactrocera oleae. Each of the two genes, named Adh1 and Adh2, consists of three exons and two introns for a total length of 1981 and 988 nucleotides, respectively. Their deduced amino acid sequences of 257 and 258 residues exhibit a 77% identity and display the characteristics of the insect ADH enzymes, which belong to the short-chain dehydrogenases/reductases family. The Adh genes of B. oleae are compared to the two genes of the Mediterranean fly, Ceratitis capitata, the only other species of the Tephritidae family in which the Adh genes have been studied. On the basis of amino acid divergence the four genes form two clusters each containing one gene from each species, as expected if there was one duplication event before speciation. On the basis of nucleotide sequence the four sequences form two clusters each containing the two sequences from the same species, as expected if there was a separate duplication event in each species. To help decide between the two alternatives, we compared at both the amino acid and DNA level the Adh genes of five Drosophila species that are known to carry two such genes and observed that, with only one exception at the amino acid level, conspecific loci cluster together. We conclude that the information we have at present does not allow a firm choice between the hypothesis of a single duplication event that occurred before the split of Bactrocera and Ceratitis from their common ancestor and the hypothesis of two independent duplication events, one in each of the two genera. Received: 30 May 2000 / Accepted: 17 August 2000     

Evolution of 28S Ribosomal DNA in Chaetognaths: Duplicate Genes and Molecular Phylogeny

The chaetognaths are an extraordinarily homogeneous phylum of animals at the morphological level, with a bauplan that can be traced back to the Cambrian. Despite the attention of zoologists for over two centuries, there is little agreement on classification within the phylum. We have used a molecular biological approach to investigate the phylogeny of extant chaetognaths. A rapidly evolving expansion segment toward the 5′ end of 28S ribosomal DNA (rDNA) was amplified using the polymerase chain reaction (PCR), cloned, and sequenced from 26 chaetognath samples representing 18 species. An unusual finding was the presence of two distinct classes of 28S rDNA gene in chaetognaths; our analyses suggest these arose by a gene (or gene cluster) duplication in a common ancestor of extant chaetognaths. The two classes of chaetognath 28S rDNA have been subject to different rates of molecular evolution; we present evidence that both are expressed and functional. In phylogenetic reconstructions, the two classes of 28S rDNA yield trees that root each other; these clearly demonstrate that the Aphragmophora and Phragmophora are natural groups. Within the Aphragmophora, we find good support for the groupings denoted Solidosagitta, Parasagitta, and Pseudosagitta. The relationships between several well-supported groups within the Aphragmophora are uncertain; we suggest this reflects rapid, recent radiation during chaetognath evolution. Received: 19 March 1996 / Accepted: 5 August 1996     

Isolation of Mhc Class II DMA and DMB cDNA Sequences in a Marsupial: The Gray Short-Tailed Opossum (Monodelphis domestica)

We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the α-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified. Received: 2 March 1998 / Accepted: 2 June 1998     

Segmented Gene Conversion as a Mechanism of Correction of 18S rRNA Pseudogene Located Outside of rDNA Cluster in D. melanogaster

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction. Received: 8 August 1996 / Accepted: 7 December 1996     

Intraspecies Analysis: Comparison of ITS Sequence Data and Gene Intron Sequence Data with Breeding Data for a Worldwide Collection of Gonium pectorale

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis. Received: 9 March 1998 / Accepted: 1 June 1998     

Neutral and Nonneutral Mitochondrial Genetic Variation in Deep-Sea Clams from the Family Vesicomyidae

Nucleotide sequences at two mitochondrial genes from 57 individuals representing eight species of deep-sea clams (Vesicomyidae) were examined for variation consistent with the neutral model of molecular evolution. One gene, cytochrome oxidase subunit I (COI), deviated from the expectations of neutrality by containing an excess of intraspecific nonsynonymous polymorphism. Additionally, one species, Calyptogena kilmeri, showed a significant excess of rare polymorphism specifically at the COI locus. In contrast, a second mitochondrial gene, the large-subunit 16S ribosomal RNA gene (16S), showed little deviation from neutrality either between or within species. Together, COI and 16S show no deviation from neutral expectations by the HKA test, produce congruent phylogenetic relationships between species, and show correlated numbers of fixed differences between species and polymorphism within species. These patterns of both neutral and nonneutral evolution within the mitochondrial genome are most consistent with a model where intraspecific nonsynonymous polymorphism at COI is near neutrality. In addition to examining the forces of molecular evolution, we extend hypotheses about interspecific relationships within this family for geographical locations previously unexamined by molecular methods including habitats near the Middle Atlantic, the Aleutian Trench, and Costa Rica. Received: 10 March 1999 / Accepted: 13 September 1999     

Patterns of Gene Duplication in Lepidopteran Pheromone Binding Proteins

We have isolated and characterized cDNAs representing two distinct pheromone binding proteins (PBPs) from the gypsy moth, Lymantria dispar. We use the L. dispar protein sequences, along with other published lepidopteran PBPs, to investigate the evolutionary relationships among genes within the PBP multigene family. Our analyses suggest that the presence of two distinct PBPs in genera representing separate moth superfamilies is the result of relatively recent, independent, gene duplication events rather than a single, ancient, duplication. We discuss this result with respect to the biochemical diversification of moth PBPs. Received: 19 March 1997 / Accepted: 11 July 1997     

Molecular phylogeny of land and freshwater planarians (Tricladida, Platyhelminthes): from freshwater to land and back

The suborder Tricladida (phylum Platyhelminthes) comprises the well-known free-living flatworms, taxonomically grouped into three infraorders according to their ecology: Maricola (marine planarians), Paludicola (freshwater planarians), and Terricola (land planarians). Molecular analyses have demonstrated that the Paludicola are paraphyletic, the Terricola being the sister group of one of the three paludicolan families, the Dugesiidae. However, neither 18S rDNA nor COI based trees have been able to resolve the relationships among species of Terricola and Dugesiidae, particularly the monophyly of Terricola. Here, we present new molecular data including sequences of nuclear genes (18S rDNA, 28S rDNA) and a mitochondrial gene (COI) of a wider sample of dugesiid and terricolan species. The new sequences have been analyzed, together with those previously obtained, in independent and concatenated analyses using maximum likelihood and bayesian methods. The results show that, although some parts of the trees remain poorly resolved, they support a monophyletic origin for Terricola followed by a likely return of some species to freshwater habitats. Relationships within the monophyletic group of Dugesiidae are clearly resolved, and relationships among some terricolan subfamilies are also clearly established and point to the need for a thorough revision of Terricola taxonomy.     

Sucrose Phosphate Synthase Genes in Plants Belong to Three Different Families

We present phylogenetic analyses to demonstrate that there are three families of sucrose phosphate synthase (SPS) genes present in higher plants. Two data sets were examined, one consisting of full-length proteins and a second larger set that covered a highly conserved region including the 14-3-3 binding region and the UDPGlu active site. Analysis of both datasets showed a well supported separation of known genes into three families, designated A, B, and C. The genomic sequences of Arabidopsis thaliana include a member in each family: two genes on chromosome 5 belong to Family A, one gene on chromosome 1 to Family B, and one gene on chromosome 4 to Family C. Each of three Citrus genes belong to one of the three families. Intron/exon organization of the four Arabidopsis genes differed according to phylogenetic analysis, with members of the same family from different species having similar genomic organization of their SPS genes. The two Family A genes on Arabidopsis chromosome 5 appear to be due to a recent duplication. Analysis of published literature and ESTs indicated that functional differentiation of the families was not obvious, although B family members appear not to be expressed in roots. B family genes were cloned from two Actinidia species and southern analysis indicated the presence of a single gene family, which contrasts to the multiple members of Family A in Actinidia. Only two family C genes have been reported to date. Received: 17 April 2001 / Accepted: 27 August 2001     

The Evolutionary History of Carbamoyltransferases: A Complex Set of Paralogous Genes Was Already Present in the Last Universal Common Ancestor

Forty-four sequences of ornithine carbamoyltransferases (OTCases) and 33 sequences of aspartate carbamoyltransferases (ATCases) representing the three domains of life were multiply aligned and a phylogenetic tree was inferred from this multiple alignment. The global topology of the composite rooted tree (each enzyme family being used as an outgroup to root the other one) suggests that present-day genes are derived from paralogous ancestral genes which were already of the same size and argues against a mechanism of fusion of independent modules. A closer observation of the detailed topology shows that this tree could not be used to assess the actual order of organismal descent. Indeed, this tree displays a complex topology for many prokaryotic sequences, with polyphyly for Bacteria in both enzyme trees and for the Archaea in the OTCase tree. Moreover, representatives of the two prokaryotic Domains are found to be interspersed in various combinations in both enzyme trees. This complexity may be explained by assuming the occurrence of two subfamilies in the OTCase tree (OTC α and OTC β) and two other ones in the ATCase tree (ATC I and ATC II). These subfamilies could have arisen from duplication and selective losses of some differentiated copies during the successive speciations. We suggest that Archaea and Eukaryotes share a common ancestor in which the ancestral copies giving the present-day ATC II/OTC β combinations were present, whereas Bacteria comprise two classes: one containing the ATC II/OTC α combination and the other harboring the ATC I/OTC β combination. Moreover, multiple horizontal gene transfers could have occurred rather recently amongst prokaryotes. Whichever the actual history of carbamoyltransferases, our data suggest that the last common ancestor to all extant life possessed differentiated copies of genes coding for both carbamoyltransferases, indicating it as a rather sophisticated organism.     

Accelerated Evolution of Cytochrome b in Simian Primates: Adaptive Evolution in Concert with Other Mitochondrial Proteins?

We have sequenced the cytochrome b gene of Horsfield's tarsier, Tarsius bancanus, to complete a data set of sequences for this gene from representatives of each primate infraorder. These primate cytochrome b sequences were combined with those from representatives of three other mammalian orders (cat, whale, and rat) in an analysis of relative evolutionary rates. The nonsynonymous nucleotide substitution rate of the cytochrome b gene has increased approximately twofold along lineages leading to simian primates compared to that of the tarsier and other primate and nonprimate mammalian species. However, the rate of transversional substitutions at fourfold degenerate sites has remained uniform among all lineages. This increase in the evolutionary rate of cytochrome b is similar in character and magnitude to that described previously for the cytochrome c oxidase subunit II gene. We propose that the evolutionary rate increase observed for cytochrome b and cytochrome c oxidase subunit II may underlie an episode of coadaptive evolution of these two proteins in the mitochondria of simian primates. Received: 15 December 1997 / Accepted: 24 February 1998     

The Presence of GSI-Like Genes in Higher Plants: Support for the Paralogous Evolution of GSI and GSII Genes

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution. Received: 4 May 1999 / Accepted: 17 September 1999     

Phylogenetic Position of the Phacotaceae Within the Chlamydophyceae as Revealed by Analysis of 18S rDNA and rbcL Sequences

Four genera of the Phacotaceae (Phacotus, Pteromonas, Wislouchiella, Dysmorphococcus), a family of loricated green algal flagellates within the Volvocales, were investigated by means of transmission electron microscopy and analysis of the nuclear encoded small-subunit ribosomal RNA (18S rRNA) genes and the plastid-encoded rbcL genes. Additionally, the 18S rDNA of Haematococcus pluvialis and the rbcL sequences of Chlorogonium elongatum, C. euchlorum, Dunaliella parva, Chloromonas serbinowii, Chlamydomonas radiata, and C. tetragama were determined. Analysis of ultrastructural data justified the separation of the Phacotaceae into two groups. Phacotus, Pteromonas, and Wislouchiella generally shared the following characters: egg-shaped protoplasts, a single pyrenoid with planar thylakoid double-lamellae, three-layered lorica, flagellar channels as part of the central lorica layer, mitochondria located in the central cytoplasm, lorica development that occurs in mucilaginous zoosporangia that are to be lysed, and no acid-resistant cell walls. Dysmorphococcus was clearly different in each of the characters mentioned. Direct comparison of sequences of Phacotus lenticularis, Pteromonas sp., Pteromonas protracta, and Wislouchiella planctonica revealed DNA sequence homologies of ≥98.0% within the 18S gene and 93.9% within the rbcL gene. D. globosus was quite different from these species, with a maximum of 92.9% homology in the 18S rRNA and ≤86.6% in the rbcL gene. It showed major similarities to the 18S rDNA of Dunaliella salina, with 95.3%, and to the rbcL sequence of Chlamydomonas tetragama, with 90.3% sequence homology. Additionally, the Phacotaceae sensu stricto exclusively shared 10 (rbcL: 4) characters which were present neither in other Chlamydomonadales nor in Dysmorphococcus globosus. Different phylogenetic analysis methods confirmed the hypothesis that the Phacotaceae are polyphyletic. The Phacotaceae sensu stricto form a stable cluster with affinities to the Dunaliellaes and possibly Haematococcus pluvialis. Dysmorphococcus globosus represented an independent lineage that is possibly related to Chlamydomonas moewusii and C. tetragama. Received: 9 June 1997 / Accepted: 17 October 1997     

Gene Conversion Drives Within Genic Sequences: Concerted Evolution of Ribosomal RNA Genes in Bacteria and Archaea

Multiple copies of a given ribosomal RNA gene family undergo concerted evolution such that sequences of all gene copies are virtually identical within a species although they diverge normally between species. In eukaryotes, gene conversion and unequal crossing over are the proposed mechanisms for concerted evolution of tandemly repeated sequences, whereas dispersed genes are homogenized by gene conversion. However, the homogenization mechanisms for multiple-copy, normally dispersed, prokaryotic rRNA genes are not well understood. Here we compared the sequences of multiple paralogous rRNA genes within a genome in 12 prokaryotic organisms that have multiple copies of the rRNA genes. Within a genome, putative sequence conversion tracts were found throughout the entire length of each individual rRNA genes and their immediate flanks. Individual conversion events convert only a short sequence tract, and the conversion partners can be any paralogous genes within the genome. Interestingly, the genic sequences undergo much slower divergence than their flanking sequences. Moreover, genomic context and operon organization do not affect rRNA gene homogenization. Thus, gene conversion underlies concerted evolution of bacterial rRNA genes, which normally occurs within genic sequences, and homogenization of flanking regions may result from co-conversion with the genic sequence. Received: 31 March 2000 / Accepted: 15 June 2000     

Comparisons of the Nucleotide Substitution Process Among Repetitive Segments of the α- and β-Spectrin Genes

The actin–cross-linking protein spectrin is a prominent component of the membrane cytoskeleton. Spectrin is a tetramer of two antiparallel αβ-dimers which share a unique and ancient gene structure. The α-spectrin and β-spectrin genes are composed primarily of tandemly repeated 106-amino-acid segments, each of which forms a triple α-helical coiled coil. Both the genes and the repeats themselves are homologous. The two genes are thought to be the result of a gene duplication event, and each gene is the product of duplications of the 106-amino-acid repeats. In this work we compare the process of molecular evolution across the repeated segments of the α- and β-spectrin genes. We find that the α-spectrin segments have, for the most part, evolved in a homogeneous fashion, while considerable heterogeneity is found among β-spectrin segments. Several segments with unique known functions are found to have evolved differently than the others. On the basis of heterogeneity of the evolutionary process, we suggest that at least one repeat has a unique function that has yet to be documented. We also present new statistical methods for comparing the evolutionary process between different regions of DNA sequences. Received: 27 March 1996 / Accepted: 21 October 1996     

Multiple Substitutions Affect the Phylogenetic Utility of Cytochrome b and 12S rDNA Data: Examining a Rapid Radiation in Leporid (Lagomorpha) Evolution

Partial sequences of two mitochondrial genes, the 12S ribosomal gene (739 bp) and the cytochrome b gene (672 bp), were analyzed in hopes of reconstructing the evolutionary relationships of 11 leporid species, representative of seven genera. However, partial cytochrome b sequences were of little phylogenetic value in this study. A suite of pairwise comparisons between taxa revealed that at the intergeneric level, the cytochrome b gene is saturated at synonymous coding positions due to multiple substitution events. Furthermore, variation at the nonsynonymous positions is limited, rendering the cytochrome b gene of little phylogenetic value for assessing the relationships between leporid genera. If the cytochrome b data are analyzed without accounting for these two classes of nucleotides (i.e., synonymous and nonsynonymous sites), one may incorrectly conclude that signal exists in the cytochrome b data. The mitochondrial 12S rRNA gene, on the other hand, has not experienced excessive saturation at either stem or loop positions. Phylogenies reconstructed from the 12S rDNA data support hypotheses based on fossil evidence that African rock rabbits (Pronolagus) are outside of the main leporid stock and that leporids experienced a rapid radiation. However, the molecular data suggest that this radiation event occurred in the mid-Miocene several millions of years earlier than the Pleistocene dates suggested by paleontological evidence. Received: 23 April 1998 / Accepted: 14 May 1998