Origin and Evolution of Paralogous rRNA Gene Clusters Within the Flatworm Family Dugesiidae (Platyhelminthes, Tricladida)

Analysis of the 18S rDNA sequences of five species of the family Dugesiidae (phylum Platyhelminthes, suborder Tricladida, infraorder Paludicola) and eight species belonging to families Dendrocoelidae and Planaridae and to the infraorder Maricola showed that members of the family Dugesiidae have two types of 18S rDNA genes, while the rest of the species have only one. The duplication event also affected the ITS-1, 5.8S, ITS-2 region and probably the 28S gene. The mean divergence value between the type I and the type II sequences is 9% and type II 18S rDNA genes are evolving 2.3 times more rapidly than type I. The evolutionary rates of type I and type II genes were calibrated from biogeographical data, and an approximate date for the duplication event of 80–120 million years ago was calculated. The type II gene was shown, by RT-PCR, to be transcribed in adult individuals of Schmidtea polychroa, though at very low levels. This result, together with the fact that most of the functionally important positions for small-subunit rRNA in prokaryotes have been conserved, indicates that the type II gene is probably functional. Received: 24 March 1998 / Accepted: 17 March 1999     

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     

Phylogenetic Position of Mammoth and Steller's Sea Cow Within Tethytheria Demonstrated by Mitochondrial DNA Sequences

Here we report DNA sequences from mitochondrial cytochrome b gene segments (1,005 base pairs per species) for the extinct woolly mammoth (Mammuthus primigenius) and Steller's sea cow (Hydrodamalis gigas) and the extant Asian elephant (Elephas maximus), the Western Indian manatee (Trichechus manatus), and the hyrax (Procavia capensis). These molecular data have allowed us to construct the phylogeny for the Tethytheria. Our molecular data resolve the trichotomy between the two species of living elephants and the mammoth and confirm that the mammoth was more closely related to the Asian elephant than to the African elephant. Our data also suggest that the sea cow–dugong divergence was likely as ancient as the dugong–manatee split, and it appears to have been much earlier (22 million years ago) than had been previously estimated (4–8 million years ago) by immunological comparison. Received: 8 August 1996 / Accepted: 30 September 1996     

The phylogenetic affinities of the chaetognaths: a molecular analysis

The chaetognaths, or arrowworms, constitute a small and enigmatic phylum of marine invertebrates whose phylogenetic affinities have long been uncertain. A popular hypothesis is that the chaetognaths are the sister group of the major deuterostome phyla: chordates, hemichordates, and echinoderms. Here we attempt to determine the affinities of the chaetognaths by using molecular sequence data. We describe the isolation and nucleotide sequence determination of 18S ribosomal DNA from one species of chaetognath and one acanthocephalan. Extensive phylogenetic analyses employing a suite of phylogenetic reconstruction methods (maximum parsimony, maximum likelihood, evolutionary parsimony, and two distance methods) suggest that the hypothesized relationship between chaetognaths and the deuterostomes is incorrect. In contrast, we propose that the lineage leading to the chaetognaths arose prior to the advent of the coelomate metazoa.      

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     

Use of a deviant mitochondrial genetic code in yellow-green algae as a landmark for segregating members within the phylum

Several algae that were previously classified in the phylum Xanthophyta (yellow-green algae) were assigned in 1971 to a new phylum, Eustigmatophyta. It was anticipated that the number of algae reclassified to Eustigmatophyta would increase. However, due to the fact that the morphological characteristics that segregate eustigmatophytes from other closely related algae can be only obtained through laborious electron microscopic techniques, the number of members in this phylum have increased rather slowly. We attempted, therefore, to segregate two closely related groups of algae, eustigmatophytes and yellow-green algae, on the basis of a molecular phylogenetic tree as a means of providing an alternative method of distinguishing these phyla. We analyzed the mitochondrial cytochrome oxidase subunit I (COXI) gene sequences of eight algae classified as xanthophyceans and found that six manifested the expected deviant genetic code where AUA codes for methionine (AUA/Met), but not for isoleucine (AUA/Ile) as in the universal genetic code. The other two, Monodus sp. (CCMP 505) and Ophiocytium majus (CCAP 855/1), which were presumed to be yellow-green algae, and all the examined eustigmatophytes utilized AUA for Ile. In addition, the phylogenetic tree of COXI gene sequences showed that the six yellow-green algae bearing the AUA/Met deviant code composed a tight clade with a bootstrap value of 100%. The phylogenetic tree of the corresponding sequences from Monodus sp. and Ophiocytium majus and the eustigmatophytes also composed a tight cluster, but with a bootstrap value of 92%. These results strongly suggest that two previously classified members of yellow-green algae belong to the phylum Eustigmatophyta. Therefore, examination of the mitochondrial genetic code in algae appears to be a potentially very useful genetic marker for classifying these organisms, especially when it is considered with the results obtained through a molecular phylogenetic tree. Received: 14 December 1996 / Accepted: 3 April 1997     

Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae

This study provides a phylogenetic/comparative approach to deciphering the processes underlying the evolution of plastid rRNA genes in genomes under relaxed functional constraints. Nonphotosynthetic green algal taxa that belong to two distinct classes, Chlorophyceae (Polytoma) and Trebouxiophyceae (Prototheca), were investigated. Similar to the situation described previously for plastid 16S rRNA genes in nonphotosynthetic land plants, nucleotide substitution levels, extent of structural variations, and percentage AT values are increased in nonphotosynthetic green algae compared to their closest photosynthetic relatives. However, the mutational processes appear to be different in many respects. First, with the increase in AT content, more transversions are noted in Polytoma and holoparasite angiosperms, while more transitions characterize the evolution of the 16S rDNA sequences in Prototheca. Second, although structural variations do accumulate in both Polytoma and Prototheca (as well as holoparasitic plastid 16S rRNAs), insertions as large as 1.6 kb characterize the plastid 16S rRNA genes in the former, whereas significantly smaller indels (not exceeding 24 bp) seem to be more prevalent in the latter group. The differences in evolutionary rates and patterns within and between lineages might be due to mutations in replication/repair-related genes; slipped-strand mispairing is likely the mechanism responsible for the expansion of insertions in Polytoma plastid 16S rRNA genes. Received: 29 December 2000 / Accepted: 18 May 2001     

The Hox gene complement of a pelagic chaetognath, Flaccisagitta enflata

Chaetognaths are transparent marine animals that are ubiquitous and abundant members of oceanic zooplanktonic communities. Their phylogenetic position within the Metazoa, however, has remained obscure since their discovery. Morphology and embryology have traditionally allied chaetognaths with deuterostomes, but molecular evidence suggests otherwise. Two recent multigene expressed sequence tag (EST) molecular phylogenomic studies suggest that chaetognaths are either sister to the Lophotrochozoa (Matus et al. 2006) or to all protostomes (Marlétaz et al. 2006). We have isolated eight Hox genes, one Parahox gene, and Mox, a related homeodomain gene, from the pelagic chaetognath, Flaccisagitta enflata. Although chaetognath central class Hox genes lack the Lox5 or "spiralian" parapeptide, a diagnostic amino-acid motif that has been utilized previously to assign lophotrochozoan affinity, they do possess a central class Hox gene that has a partial "Ubd-A peptide" found in both ecdysozoan and lophotrochozoan Ubx/Abd-A/Lox2/Lox4 genes. Additionally, we report the presence of two distinct chaetognath posterior Hox genes that possess both ecdysozoan and lophotrochozoan signature amino-acid motifs. The phylogenetic position of chaetognaths, as well as the evolution of the Hox cluster, is discussed in light of these data.     

Phylomitogenomic analyses strongly support the sister relationship of the Chaetognatha and Protostomia

The phylum Chaetognatha (arrow worms) comprises a group of small marine predators that constitute a critical component of the zooplankton community throughout the world's oceans. Various phylogenetic affiliations have been proposed for the Chaetognatha, for which there are at least nine possible phylogenetic positions. Resolving the phylogenetic position of the chaetognaths is a key in understanding the fundamental developmental features of bilaterians. In comparison with the typical gene content of metazoan mitogenomes, two protein‐coding genes (atp6 and atp8) are absent from all chaetognaths. The two mitogenomes sequenced from Sagitta crassa and Zonosagitta nagae in this study nevertheless contain two and four tRNA genes, respectively, in contrast to those of the other five chaetognaths reported where only one tRNA gene (trnMet) is present, thus invalidating the view that all chaetognath mitogenomes have a single tRNA gene. A conserved major gene order shared by all chaetognaths could be partially identified in many protostome mitogenomes, but not in any ancestral mitogenome gene arrangement of the four deuterostome groups. Phylogenetic analysis of the deduced amino acid sequences of protein‐coding genes from 85 mitogenomes of 19 groups suggests the Chaetognatha to be a sister group to the protostomes, a result consistent with evidences from the developmental pattern and other molecular analyses.     

Molecular Characterization of the Principal Symbiotic Bacteria of the Weevil Sitophilus oryzae: A Peculiar G + C Content of an Endocytobiotic DNA

The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae. Received: 8 September 1997 / Accepted: 24 October 1997     

Group I Introns from Zygomycota: Evolutionary Implications for the Fungal IC1 Intron Subgroup

The origins of fungal group I introns within nuclear small-subunit (nSSU) rDNA are enigmatic. This is partly because they have never been reported in basal fungal phyla (Zygomycota and Chytridiomycota), which are hypothesized to be ancestral to derived phyla (Ascomycota and Basidiomycota). Here we report group I introns from the nSSU rDNA of two zygomycete fungi, Zoophagus insidians (Zoopagales) and Coemansia mojavensis (Kickxellales). Secondary structure analyses predicted that both introns belong to the IC1 subgroup and that they are distantly related to each other, which is also suggested by different insertion sites. Molecular phylogenetic analyses indicated that the IC1 intron of Z. insidians is closely related to the IC1 intron inserted in the LSU rDNA of the basidiomycete fungus Clavicorona taxophila, which strongly suggests interphylum horizontal transfer. The IC1 intron of C. mojavensis has a low phylogenetic affinity to other fungal IC1 introns inserted into site 943 of nSSU rDNA (relative to E. coli 16S rDNA). It is noteworthy that this intron contains a putative ORF containing a His–Cys box motif in the antisense strand, a hallmark for nuclear-encoded homing endonucleases. Overall, molecular phylogenetic analyses do not support the placement of these two introns in basal fungal IC1 intron lineages. This result leads to the suggestion that fungal IC1 introns might have invaded or been transferred laterally after the divergence of the four major fungal phyla. Received: 8 February 2001 / Accepted: 1 November 2001     

Phylogenetic Position of the Hexactinellida Within the Phylum Porifera Based on the Amino Acid Sequence of the Protein Kinase C from Rhabdocalyptus dawsoni

Recent analyses of genes encoding proteins typical for multicellularity, especially adhesion molecules and receptors, favor the conclusion that all metazoan phyla, including the phylum Porifera (sponges), are of monophyletic origin. However, none of these data includes cDNA encoding a protein from the sponge class Hexactinellida. We have now isolated and characterized the cDNA encoding a protein kinase C, belonging to the C subfamily (cPKC), from the hexactinellid sponge Rhabdocalyptus dawsoni. The two conserved regions, the regulatory part with the pseudosubstrate site, the two zinc fingers, and the C2 domain, as well as the catalytic domain were used for phylogenetic analyses. Sequence alignment and construction of a phylogenetic tree from the catalytic domains revealed that the yeast Saccharomyces cerevisiae and the protozoan Trypanosoma brucei are at the base of the tree, while the hexactinellid R. dawsoni branches off first among the metazoan sequences; the other two classes of the Porifera, the Calcarea (the sequence from Sycon raphanus was used) and the Demospongiae (sequences from Geodia cydonium and Suberites domuncula were used), branch off later. The statistically robust tree also shows that the two cPKC sequences from the higher invertebrates Drosophila melanogaster and Lytechinus pictus are most closely related to the calcareous sponge. This finding was also confirmed by comparing the regulatory part of the kinase gene. We suggest, that (i) within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor to the Calcarea and the Demospongiae, which both appeared later, and (ii) the higher invertebrates are more closely related to the calcareous sponges. Received: 6 August 1997 / Accepted: 24 October 1997     

Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleractinian corals

Relationships among families and suborders of scleractinian corals are poorly understood because of difficulties 1) in making inferences about the evolution of the morphological characters used in coral taxonomy and 2) in interpreting their 240-million-year fossil record. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 16S ribosomal gene from taxa of 14 families of corals and the use of this gene segment in a phylogenetic analysis of relationships within the order. We show that sequences obtained from scleractinians are homologous to other metazoan 16S ribosomal sequences and fall into two distinct clades defined by size of the amplified gene product. Comparisons of sequences from the two clades demonstrate that both sets of sequences are evolving under similar evolutionary constraints: they do not differ in nucleotide composition, numbers of transition and transversion substitutions, spatial patterns of substitutions, or in rates of divergence. The characteristics and patterns observed in these sequences as well as the secondary structures, are similar to those observed in mt 16S ribosomal DNA sequences from other taxa. Phylogenetic analysis of these sequences shows that they are useful for evaluating relationships within the order. The hypothesis generated from this analysis differs from traditional hypotheses for evolutionary relationships among the Scleractinia and suggests that a reevaluation of evolutionary affinities in the order is needed. Received: 4 September 1996 / Accepted: 7 April 1997     

Curiously Modern DNA for a ``250 Million-Year-Old' Bacterium

Studies of ancient DNA have attracted considerable attention in scientific journals and the popular press. Several of the more extreme claims for ancient DNA have been questioned on biochemical grounds (i.e., DNA surviving longer than expected) and evolutionary grounds (i.e., nucleotide substitution patterns not matching theoretical expectations for ancient DNA). A recent letter to Nature from Vreeland et al. (2000), however, tops all others with respect to age and condition of the specimen. These researchers extracted and cultured a bacterium from an inclusion body from what they claim is a 250 million-year (Myr)-old salt crystal. If substantiated, this observation could fundamentally alter views about bacterial physiology, ecology and evolution. Here we report on molecular evolutionary analyses of the 16S rDNA from this specimen. We find that 2-9-3 differs from a modern halophile, Salibacillus marismortui, by just 3 unambiguous bp in 16S rDNA, versus the ∼59 bp that would be expected if these bacteria evolved at the same rate as other bacteria. We show, using a Poisson distribution, that unless it can be shown that S. marismortui evolves 5 to 10 times more slowly than other bacteria for which 16S rDNA substitution rates have been established, Vreeland et al.'s claim would be rejected at the 0.05 level. Also, a molecular clock test and a relative rates test fail to substantiate Vreeland et al.'s claim that strain 2-9-3 is a 250-Myr-old bacterium. The report of Vreeland et al. thus falls into a long series of suspect ancient DNA studies. Received: 12 April 2001 / Accepted: 9 June 2001     

Systematics of Chaetognatha under the light of molecular data, using duplicated ribosomal 18S DNA sequences

While the phylogenetic position of Chaetognatha has became central to the question of early bilaterian evolution, the internal systematics of the phylum are still not clear. The phylogenetic relationships of the chaetognaths were investigated using newly obtained small subunit ribosomal RNA nuclear 18S (SSU rRNA) sequences from 16 species together with 3 sequences available in GenBank. As previously shown with the large subunit ribosomal RNA 28S gene, two classes of Chaetognatha SSU rRNA gene can be identified, suggesting a duplication of the whole ribosomal cluster; allowing the rooting of one class of genes by another in phylogenetic analyses. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the molecular data, and statistical tests showed (1) that there are three main monophyletic groups: Sagittidae/Krohnittidae, Spadellidae/Pterosagittidae, and Eukrohniidae/Heterokrohniidae, (2) that the group of Aphragmophora without Pterosagittidae (Sagittidae/Krohnittidae) is monophyletic, (3) the Spadellidae/Pterosagittidae and Eukrohniidae/Heterokrohniidae families are very likely clustered, (4) the Krohnittidae and Pterosagittidae groups should no longer be considered as families as they are included in other groups designated as families, (5) suborder Ctenodontina is not monophyletic and the Flabellodontina should no longer be considered as a suborder, and (6) the Syngonata/Chorismogonata and the Monophragmophora/Biphragmophora hypotheses are rejected. Such conclusions are considered in the light of morphological characters, several of which are shown to be prone to homoplasy.     

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