Characterization of the Mitochondrial Proteome in the Ctenophore Mnemiopsis leidyi Using MitoPredictor.

Mitochondrial proteomes have been experimentally characterized for only a handful of animal species. However, the increasing availability of genomic and transcriptomic data allows one to infer mitochondrial proteins using computational tools. MitoPredictor is a novel random forest classifier, which utilizes orthology search, mitochondrial targeting signal (MTS) identification, and protein domain content to infer mitochondrial proteins in animals. MitoPredictor's output also includes an easy-to-use R Shiny applet for the visualization and analysis of the results. In this article, we provide a guide for predicting and analyzing the mitochondrial proteome of the ctenophore Mnemiopsis leidyi using MitoPredictor.

Phylomitogenomics bolsters the high-level classification of Demospongiae (phylum Porifera).

Class Demospongiae is the largest in the phylum Porifera (Sponges) and encompasses nearly 8,000 accepted species in three subclasses: Keratosa, Verongimorpha, and Heteroscleromorpha. Subclass Heteroscleromorpha contains ∼90% of demosponge species and is subdivided into 17 orders. The higher level classification of demosponges underwent major revision as the result of nearly three decades of molecular studies. However, because most of the previous molecular work only utilized partial data from a small number of nuclear and mitochondrial (mt) genes, this classification scheme needs to be tested by larger datasets. Here we compiled a mt dataset for 136 demosponge species-including 64 complete or nearly complete and six partial mt-genome sequences determined or assembled for this study-and used it to test phylogenetic relationships among Demospongiae in general and Heteroscleromorpha in particular. We also investigated the phylogenetic position of Myceliospongia araneosa, a highly unusual demosponge without spicules and spongin fibers, currently classified as Demospongiae incertae sedis, for which molecular data were not available. Our results support the previously inferred sister-group relationship between Heteroscleromorpha and Keratosa + Verongimorpha and suggest five main clades within Heteroscleromorpha: Clade C0 composed of order Haplosclerida; Clade C1 composed of Scopalinida, Sphaerocladina, and Spongillida; Clade C2 composed of Axinellida, Biemnida, Bubarida; Clade C3 composed of Tetractinellida; and Clade C4 composed of Agelasida, Clionaida, Desmacellida, Merliida, Suberitida, Poecilosclerida, Polymastiida, and Tethyida. The inferred relationships among these clades were (C0(C1(C2(C3+C4)))). Analysis of molecular data from M. araneosa placed it in the C3 clade as a sister taxon to the highly skeletonized tetractinellids Microscleroderma sp. and Leiodermatium sp. Molecular clock analysis dated divergences among the major clades in Heteroscleromorpha from the Cambrian to the Early Silurian, the origins of most heteroscleromorph orders in the middle Paleozoic, and the most basal splits within these orders around the Paleozoic to Mesozoic transition. Overall, the results of this study are mostly congruent with the accepted classification of Heteroscleromorpha, but add temporal perspective and new resolution to phylogenetic relationships within this subclass.

Large dataset of octocoral mitochondrial genomes provides new insights into mt-mutS evolution and function.

All studied octocoral mitochondrial genomes (mt-genomes) contain a homologue of the Escherichia coli mutS gene, a member of a gene family encoding proteins involved in DNA mismatch repair, other types of DNA repair, meiotic recombination, and other functions. Although mutS homologues are found in all domains of life, as well as viruses, octocoral mt-mutS is the only such gene found in an organellar genome. While the function of mtMutS is not known, its domain architecture, conserved sequence, and presence of several characteristic residues suggest its involvement in mitochondrial DNA repair. This inference is supported by exceptionally low rates of mt-sequence evolution observed in octocorals. Previous studies of mt-mutS have been limited by the small number of octocoral mt-genomes available. We utilized sequence-capture data from the recent Quattrini et al. 2020 study [Nature Ecology & Evolution 4:1531-1538] to assemble complete mt-genomes for 94 species of octocorals. Combined with sequences publicly available in GenBank, this resulted in a dataset of 184 complete mt-genomes, which we used to re-analyze the conservation and evolution of mt-mutS. In our analysis, we discovered the first case of mt-mutS loss among octocorals in one of the two Pseudoanthomastus spp. assembled from Quattrini et al. data. This species displayed accelerated rate and changed patterns of nucleotide substitutions in mt-genome, which we argue provide additional evidence for the role of mtMutS in DNA repair. In addition, we found accelerated mt-sequence evolution in the presence of mt-mutS in several octocoral lineages. This accelerated evolution did not appear to be the result of relaxed selection pressure and did not entail changes in patterns of nucleotide substitutions. Overall, our results support previously reported patterns of conservation in mt-mutS and suggest that mtMutS is involved in DNA repair in octocoral mitochondria. They also indicate that the presence of mt-mutS contributes to, but does not fully explain, the low rates of sequence evolution in octocorals.

Multiple Losses of MSH1, Gain of mtMutS, and Other Changes in the MutS Family of DNA Repair Proteins in Animals.

MutS is a key component of the mismatch repair (MMR) pathway. Members of the MutS protein family are present in prokaryotes, eukaryotes, and viruses. Six MutS homologs (MSH1-6) have been identified in yeast, of which three function in nuclear MMR, while MSH1 functions in mitochondrial DNA repair. MSH proteins are believed to be well conserved in animals, except for MSH1-which is thought to be lost. Two intriguing exceptions to this general picture have been found, both in the class Anthozoa within the phylum Cnidaria. First, an ortholog of the yeast-MSH1 was reported in one hexacoral species. Second, a MutS homolog (mtMutS) has been found in the mitochondrial genome of all octocorals. To understand the origin and potential functional implications of these exceptions, we investigated the evolution of the MutS family both in Cnidaria and in animals in general. Our study confirmed the acquisition of octocoral mtMutS by horizontal gene transfer from a giant virus. Surprisingly, we identified MSH1 in all hexacorals and several sponges and placozoans. By contrast, MSH1 orthologs were lacking in other cnidarians, ctenophores, and bilaterian animals. Furthermore, while we identified MSH2 and MSH6 in nearly all animals, MSH4, MSH5, and, especially, MSH3 were missing in multiple species. Overall, our analysis revealed a dynamic evolution of the MutS family in animals, with multiple losses of MSH1, MSH3, some losses of MSH4 and MSH5, and a gain of the octocoral mtMutS. We propose that octocoral mtMutS functionally replaced MSH1 that was present in the common ancestor of Anthozoa.

Causes and consequences of mitochondrial proteome size variation in animals.

Despite a conserved set of core mitochondrial functions, animal mitochondrial proteomes show a large variation in size. We analyzed putative mechanisms behind and functional significance of this variation by performing comparative analysis of the experimentally-verified mitochondrial proteomes of four bilaterian animals (human, mouse, Caenorhabditis elegans, and Drosophila melanogaster) and two non-animal outgroups (Acanthamoeba castellanii and Saccharomyces cerevisiae). We found that of several factors affecting mitochondrial proteome size, evolution of novel mitochondrial proteins in mammals and loss of ancestral proteins in protostomes were the main contributors. Interestingly, the gain and loss of the N-terminal mitochondrial targeting signal was not a major factor in the proteome size evolution.

MMPdb and MitoPredictor: Tools for facilitating comparative analysis of animal mitochondrial proteomes.

Data on experimentally-characterized animal mitochondrial proteomes (mt-proteomes) are limited to a few model organisms and are scattered across multiple databases, impeding a comparative analysis. We developed two resources to address these problems. First, we re-analyzed proteomic data from six species with experimentally characterized mt-proteomes: animals (Homo sapiens, Mus musculus, Caenorhabditis elegans, and Drosophila melanogaster), and outgroups (Acanthamoeba castellanii and Saccharomyces cerevisiae) and created the Metazoan Mitochondrial Proteome Database (MMPdb) to host the results. Second, we developed a novel pipeline, "MitoPredictor" that uses a Random Forest classifier to infer mitochondrial localization of proteins based on orthology, mitochondrial targeting signal prediction, and protein domain analyses. Both tools generate an R Shiny applet that can be used to visualize and interact with the results and can be used on a personal computer. MMPdb is also available online at https://mmpdb.eeob.iastate.edu/.

Characterization of mitochondrial proteomes of nonbilaterian animals.

Mitochondria require ~1,500 proteins for their maintenance and proper functionality, which constitute the mitochondrial proteome (mt-proteome). Although a few of these proteins, mostly subunits of the electron transport chain complexes, are encoded in mitochondrial DNA (mtDNA), the vast majority are encoded in the nuclear genome and imported to the organelle. Previous studies have shown a continuous and complex evolution of mt-proteome among eukaryotes. However, there was less attention paid to mt-proteome evolution within Metazoa, presumably because animal mtDNA and, by extension, animal mitochondria are often considered to be uniform. In this analysis, two bioinformatic approaches (Orthologue-detection and Mitochondrial Targeting Sequence prediction) were used to identify mt-proteins in 23 species from four nonbilaterian phyla: Cnidaria, Ctenophora, Placozoa, and Porifera, as well as two choanoflagellates, the closest animal relatives. Our results revealed a large variation in mt-proteome in nonbilaterian animals in size and composition. Myxozoans, highly reduced cnidarian parasites, possessed the smallest inferred mitochondrial proteomes, while calcareous sponges possessed the largest. About 513 mitochondrial orthologous groups were present in all nonbilaterian phyla and human. Interestingly, 42 human mitochondrial proteins were not identified in any nonbilaterian species studied and represent putative innovations along the bilaterian branch. Several of these proteins were involved in apoptosis and innate immunity, two processes known to evolve within Metazoa. Conversely, several proteins identified as mitochondrial in nonbilaterian phyla and animal outgroups were absent in human, representing cases of possible loss. Finally, a few human cytosolic proteins, such as histones and cytosolic ribosomal proteins, were predicted to be targeted to mitochondria in nonbilaterian animals. Overall, our analysis provides the first step in characterization of mt-proteomes in nonbilaterian animals and understanding evolution of animal mt-proteome. © 2018 IUBMB Life, 70(12):1289-1301, 2018.

Transcriptome sequencing and delimitation of sympatric Oscarella species (O. carmela and O. pearsei sp. nov) from California, USA.

The homoscleromorph sponge Oscarella carmela, first described from central California, USA is shown to represent two superficially similar but both morphologically and phylogenetically distinct species that are co-distributed. We here describe a new species as Oscarella pearsei, sp. nov. and re-describe Oscarella carmela; the original description was based upon material from both species. Further, we correct the identification of published genomic/transcriptomic resources that were originally attributed to O. carmela, and present new Illumina-sequenced transcriptome assemblies for each of these species, and the mitochondrial genome sequence for O. pearsei sp. nov. Using SSU and LSU ribosomal DNA and the mitochondrial genome, we report the phylogenetic relationships of these species relative to other Oscarella species, and find strong support for the placement of O. pearsei sp. nov. in a distinct clade within genus Oscarella defined by the presence of spherulous cells that contain paracrystalline inclusions; O. carmela lacks this cell type. Oscarella pearsei sp. nov and O. carmela can be tentatively distinguished based upon gross morphological differences such as color, surface texture and extent of mucus production, but can be more reliably identified using mitochondrial and nuclear barcode sequencing, ultrastructural characteristics of cells in the mesohyl, and the morphology of the follicle epithelium which surrounds the developing embryo in reproductively active individuals.

Integrative taxonomic re-description of Halisarca magellanica and description of a new species of Halisarca (Porifera, Demospongiae) from Chilean Patagonia.

A series of recent expeditions in fjords and canals of Southern Chilean Patagonia allowed the re-collection of Halisarca magellanica Topsent, 1901 and the discovery of a new species, Halisarca desqueyrouxae sp. nov. The material studied was collected at depths ranging from 3 to 30 m at latitudes comprised between 42° and 49°S. Both species share the same habitat and show a morphological plasticity, but differ in their colour. Halisarca magellanica is bright pink to whitish with three morphs whereas H. desqueyrouxae sp. nov. is light brown to beige with two morphs. An extensive investigation in TEM and SEM reveals several differences among cell types with inclusions between both species. Three distinct spherulous cells occur. Type 1 is shared by both species, Type 2 is occasional in H. magellanica but absent from H. desqueyrouxae sp. nov. Type 3 is rare in H. magellanica and occurs abundantly in half of the specimens of H. desqueyrouxae sp. nov. Granular cells are shared by both species but do not occur in all specimens. Microgranular cells are characteristic of H. magellanica. Both species also clearly differ by their endobiotic bacteria. Phylogenetic analysis of cox1 sequences places H. magellanica as a sister group to all other previously published Halisarca species sequences (9.1-9.7% difference) except H. harmelini, while H. desqueyrouxae sp. nov. is placed as a sister group to H. dujardini (2.3% difference).

Animal Mitochondrial DNA as We Do Not Know It: mt-Genome Organization and Evolution in Nonbilaterian Lineages.

Animal mitochondrial DNA (mtDNA) is commonly described as a small, circular molecule that is conserved in size, gene content, and organization. Data collected in the last decade have challenged this view by revealing considerable diversity in animal mitochondrial genome organization. Much of this diversity has been found in nonbilaterian animals (phyla Cnidaria, Ctenophora, Placozoa, and Porifera), which, from a phylogenetic perspective, form the main branches of the animal tree along with Bilateria. Within these groups, mt-genomes are characterized by varying numbers of both linear and circular chromosomes, extra genes (e.g. atp9, polB, tatC), large variation in the number of encoded mitochondrial transfer RNAs (tRNAs) (0-25), at least seven different genetic codes, presence/absence of introns, tRNA and mRNA editing, fragmented ribosomal RNA genes, translational frameshifting, highly variable substitution rates, and a large range of genome sizes. This newly discovered diversity allows a better understanding of the evolutionary plasticity and conservation of animal mtDNA and provides insights into the molecular and evolutionary mechanisms shaping mitochondrial genomes.

Extensive Mitochondrial mRNA Editing and Unusual Mitochondrial Genome Organization in Calcaronean Sponges.

One of the unusual features of DNA-containing organelles in general and mitochondria in particular is the frequent occurrence of RNA editing [1]. The term "RNA editing" refers to a variety of mechanistically unrelated biochemical processes that alter RNA sequence during or after transcription [2]. The editing can be insertional, deletional, or substitutional and has been found in all major types of RNAs [3, 4]. Although mitochondrial mRNA editing is widespread in some eukaryotic lineages [5-7], it is rare in animals, with reported cases limited both in their scope and in phylogenetic distribution [8-11] (see also [12]). While analyzing genomic data from calcaronean sponges Sycon ciliatum and Leucosolenia complicata, we were perplexed by the lack of recognizable mitochondrial coding sequences. Comparison of genomic and transcriptomic data from these species revealed the presence of mitochondrial cryptogenes whose transcripts undergo extensive editing. This editing consisted of single or double uridylate (U) insertions in pre-existing short poly(U) tracts. Subsequent analysis revealed the presence of similar editing in Sycon coactum and the loss of editing in Petrobiona massiliana, a hypercalcified calcaronean sponge. In addition, mitochondrial genomes of at least some calcaronean sponges were found to have a highly unusual architecture, with nearly all genes located on individual and likely linear chromosomes. Phylogenetic analysis of mitochondrial coding sequences revealed accelerated rates of sequence evolution in this group. The latter observation presents a challenge for the mutational-hazard hypothesis [13], which posits that mRNA editing should not occur in lineages with an elevated mutation rate.

Ancestral state reconstruction infers phytopathogenic origins of sooty blotch and flyspeck fungi on apple.

Members of the sooty blotch and flyspeck (SBFS) complex are epiphytic fungi in the Ascomycota that cause economically damaging blemishes of apples worldwide. SBFS fungi are polyphyletic, but approx. 96% of SBFS species are in the Capnodiales. Evolutionary origins of SBFS fungi remain unclear, so we attempted to infer their origins by means of ancestral state reconstruction on a phylogenetic tree built utilizing genes for the nuc 28S rDNA (approx. 830 bp from near the 59 end) and the second largest subunit of RNA polymerase II (RPB2). The analyzed taxa included the well-known genera of SBFS as well as non-SBFS fungi from seven families within the Capnodiales. The non-SBFS taxa were selected based on their distinct ecological niches, including plant-parasitic and saprophytic species. The phylogenetic analyses revealed that most SBFS species in the Capnodiales are closely related to plant-parasitic fungi. Ancestral state reconstruction provided strong evidence that plant-parasitic fungi were the ancestors of the major SBFS lineages. Knowledge gained from this study may help to better understand the ecology and evolution of epiphytic fungi.

Cytonuclear Interactions in the Evolution of Animal Mitochondrial tRNA Metabolism.

The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-proteobacterial ancestor. However, variation in the complement of mitochondrial-encoded molecules involved in translation, including transfer RNAs (tRNAs), provides evidence for the ongoing evolution of mitochondrial protein synthesis. Here, we investigate the evolution of the mitochondrial translational machinery using recent genomic and transcriptomic data from animals that have experienced the loss of mt-tRNAs, including phyla Cnidaria and Ctenophora, as well as some representatives of all four classes of Porifera. We focus on four sets of mitochondrial enzymes that directly interact with tRNAs: Aminoacyl-tRNA synthetases, glutamyl-tRNA amidotransferase, tRNA(Ile) lysidine synthetase, and RNase P. Our results support the observation that the fate of nuclear-encoded mitochondrial proteins is influenced by the evolution of molecules encoded in mitochondrial DNA, but in a more complex manner than appreciated previously. The data also suggest that relaxed selection on mitochondrial translation rather than coevolution between mitochondrial and nuclear subunits is responsible for elevated rates of evolution in mitochondrial translational proteins.

Eight new mtDNA sequences of glass sponges reveal an extensive usage of +1 frameshifting in mitochondrial translation.

Three previously studied mitochondrial genomes of glass sponges (phylum Porifera, class Hexactinellida) contained single nucleotide insertions in protein coding genes inferred as sites of +1 translational frameshifting. To investigate the distribution and evolution of these sites and to help elucidate the mechanism of frameshifting, we determined eight new complete or nearly complete mtDNA sequences from glass sponges and examined individual mitochondrial genes from three others. We found nine new instances of single nucleotide insertions in these sequences and analyzed them both comparatively and phylogenetically. The base insertions appear to have been gained and lost repeatedly in hexactinellid mt protein genes, suggesting no functional significance for the frameshifting sites. A high degree of sequence conservation, the presence of unusual tRNAs, and a distinct pattern of codon usage suggest the "out-of-frame pairing" model of translational frameshifting. Additionally, we provide evidence that relaxed selection pressure on glass sponge mtDNA - possibly a result of their low growth rates and deep-water lifestyle - has allowed frameshift insertions to be tolerated for hundreds of millions of years. Our study provides the first example of a phylogenetically diverse and extensive usage of translational frameshifting in animal mitochondrial coding sequences.

The twin-arginine subunit C in Oscarella: origin, evolution, and potential functional significance.

The twin-arginine translocation (Tat) pathway is a protein transport system that moves completely folded proteins across lipid membranes. Genes encoding components of the pathway have been found in the genomes of many Bacteria, Archaea, and eukaryotic organelles including chloroplasts, plant mitochondria, and the mitochondria of many protists. However, with a single exception, Tat genes are absent from the mitochondrial genomes of all animals. The only exception comes from the homoscleromorph sponges in the family Oscarellidae, whose mitochondrial genomes encode a gene for tatC, the largest subunit of the complex. Here, we explore the origin and evolution of the mitochondrial tatC gene in Oscarellidae, and use bioinformatic approaches to evaluate its functional significance. We conclude that tatC in Homoscleromorpha sponges was likely inherited from the ancestral proto-mitochondrial genome, implying multiple independent losses of the mitochondrial Tat pathway during the evolution of opisthokonts. In addition, bioinformatic evidence suggests that tatC comprises the entire Tat pathway in Oscarellidae, and that the Rieske Fe/S protein of mitochondrial complex III is its likely substrate.

Systematics and molecular phylogeny of the family oscarellidae (homoscleromorpha) with description of two new oscarella species.

The family Oscarellidae is one of the two families in the class Homoscleromorpha (phylum Porifera) and is characterized by the absence of a skeleton and the presence of a specific mitochondrial gene, tatC. This family currently encompasses sponges in two genera: Oscarella with 17 described species and Pseudocorticium with one described species. Although sponges in this group are relatively well-studied, phylogenetic relationships among members of Oscarellidae and the validity of genus Pseudocorticium remain open questions. Here we present a phylogenetic analysis of Oscarellidae using four markers (18S rDNA, 28S rDNA, atp6, tatC), and argue that it should become a mono-generic family, with Pseudocorticium being synonymized with Oscarella, and with the transfer of Pseudocorticium jarrei to Oscarella jarrei. We show that the genus Oscarella can be subdivided into four clades, each of which is supported by either a small number of morphological characters or by molecular synapomorphies. In addition, we describe two new species of Oscarella from Norwegian fjords: O. bergenensis sp. nov. and O. nicolae sp. nov., and we compare their morphology, anatomy, and cytology with other species in this genus. Internal anatomical characters are similar in both species, but details of external morphology and particularly of cytological characters provide diagnostic features. Our study also confirms that O. lobularis and O. tuberculata are two distinct polychromic sibling species. This study highlights the difficulties of species identification in skeleton-less sponges and, more generally, in groups where morphological characters are scarce. Adopting a multi-marker approach is thus highly suitable for these groups.

The integrative taxonomic approach applied to porifera: a case study of the homoscleromorpha.

The two main scientific tasks of taxonomy are species' delineation and classification. These two tasks are often treated differently, with classification accomplished by newly-developed phylogenetic methods, often based on molecular sequences, while delimitation of species is conducted by what is often considered to be an "old-fashioned" typological approach based on morphological description. A new "integrative taxonomy" has been proposed which maintains that species delimitation should be a multidisciplinary undertaking combining several independent datasets. Here we argue that the same principle is relevant to the classification of species. In the past 20 years, we assembled various datasets based on the external morphology, anatomy, cytology, spicule shapes, geography, reproduction, genetic sequences, and metabolomics of homoscleromorph sponges. We show how we used these datasets to describe new species of homoscleromorph sponges and to elucidate their phylogenetic relationships and their phylogenetic position within the phylum Porifera.

Cnidarian phylogenetic relationships as revealed by mitogenomics.

BACKGROUND: Cnidaria (corals, sea anemones, hydroids, jellyfish) is a phylum of relatively simple aquatic animals characterized by the presence of the cnidocyst: a cell containing a giant capsular organelle with an eversible tubule (cnida). Species within Cnidaria have life cycles that involve one or both of the two distinct body forms, a typically benthic polyp, which may or may not be colonial, and a typically pelagic mostly solitary medusa. The currently accepted taxonomic scheme subdivides Cnidaria into two main assemblages: Anthozoa (Hexacorallia + Octocorallia) - cnidarians with a reproductive polyp and the absence of a medusa stage - and Medusozoa (Cubozoa, Hydrozoa, Scyphozoa, Staurozoa) - cnidarians that usually possess a reproductive medusa stage. Hypothesized relationships among these taxa greatly impact interpretations of cnidarian character evolution. RESULTS: We expanded the sampling of cnidarian mitochondrial genomes, particularly from Medusozoa, to reevaluate phylogenetic relationships within Cnidaria. Our phylogenetic analyses based on a mitochogenomic dataset support many prior hypotheses, including monophyly of Hexacorallia, Octocorallia, Medusozoa, Cubozoa, Staurozoa, Hydrozoa, Carybdeida, Chirodropida, and Hydroidolina, but reject the monophyly of Anthozoa, indicating that the Octocorallia + Medusozoa relationship is not the result of sampling bias, as proposed earlier. Further, our analyses contradict Scyphozoa [Discomedusae + Coronatae], Acraspeda [Cubozoa + Scyphozoa], as well as the hypothesis that Staurozoa is the sister group to all the other medusozoans. CONCLUSIONS: Cnidarian mitochondrial genomic data contain phylogenetic signal informative for understanding the evolutionary history of this phylum. Mitogenome-based phylogenies, which reject the monophyly of Anthozoa, provide further evidence for the polyp-first hypothesis. By rejecting the traditional Acraspeda and Scyphozoa hypotheses, these analyses suggest that the shared morphological characters in these groups are plesiomorphies, originated in the branch leading to Medusozoa. The expansion of mitogenomic data along with improvements in phylogenetic inference methods and use of additional nuclear markers will further enhance our understanding of the phylogenetic relationships and character evolution within Cnidaria.

Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code.

Sponges (phylum Porifera) are a large and ancient group of morphologically simple but ecologically important aquatic animals. Although their body plan and lifestyle are relatively uniform, sponges show extensive molecular and genetic diversity. In particular, mitochondrial genomes from three of the four previously studied classes of Porifera (Demospongiae, Hexactinellida, and Homoscleromorpha) have distinct gene contents, genome organizations, and evolutionary rates. Here, we report the mitochondrial genome of Clathrina clathrus (Calcinea, Clathrinidae), a representative of the fourth poriferan class, the Calcarea, which proves to be the most unusual. Clathrina clathrus mitochondrial DNA (mtDNA) consists of six linear chromosomes 7.6-9.4 kb in size and encodes at least 37 genes: 13 protein codings, 2 ribosomal RNAs (rRNAs), and 24 transfer RNAs (tRNAs). Protein genes include atp9, which has now been found in all major sponge lineages, but no atp8. Our analyses further reveal the presence of a novel genetic code that involves unique reassignments of the UAG codons from termination to tyrosine and of the CGN codons from arginine to glycine. Clathrina clathrus mitochondrial rRNAs are encoded in three (srRNA) and ≥6 (lrRNA) fragments distributed out of order and on several chromosomes. The encoded tRNAs contain multiple mismatches in the aminoacyl acceptor stems that are repaired posttranscriptionally by 3'-end RNA editing. Although our analysis does not resolve the phylogenetic position of calcareous sponges, likely due to their high rates of mitochondrial sequence evolution, it confirms mtDNA as a promising marker for population studies in this group. The combination of unusual mitochondrial features in C. clathrus redefines the extremes of mtDNA evolution in animals and further argues against the idea of a "typical animal mtDNA."

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles.

BACKGROUND: Haemophilus parasuis is the causative agent of Glässer's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10 mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.