The mitochondrial genomes of Amphiascoides atopus and Schizopera knabeni (Harpacticoida: Miraciidae) reveal similarities between the copepod orders Harpacticoida and Poecilostomatoida

Members of subclass Copepoda are abundant, diverse, and—as a result of their variety of ecological roles in marine and freshwater environments—important, but their phylogenetic interrelationships are unclear. Recent studies of arthropods have used gene arrangements in the mitochondrial (mt) genome to infer phylogenies, but for copepods, only seven complete mt genomes have been published. These data revealed several within-order and few among-order similarities. To increase the data available for comparisons, we sequenced the complete mt genome (13,831 base pairs) of Amphiascoides atopus and 10,649 base pairs of the mt genome of Schizopera knabeni (both in the family Miraciidae of the order Harpacticoida). Comparison of our data to those for Tigriopus japonicus (family Harpacticidae, order Harpacticoida) revealed similarities in gene arrangement among these three species that were consistent with those found within and among families of other copepod orders. Comparison of the mt genomes of our species with those known from other copepod orders revealed the arrangement of mt genes of our Harpacticoida species to be more similar to that of Sinergasilus polycolpus (order Poecilostomatoida) than to that of T. japonicus. The similarities between S. polycolpus and our species are the first to be noted across the boundaries of copepod orders and support the possibility that mt-gene arrangement might be used to infer copepod phylogenies. We also found that our two species had extremely truncated transfer RNAs and that gene overlaps occurred much more frequently than has been reported for other copepod mt genomes.     

Phylogeny and molecular evolution of the Acc1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy gene encoding plastid acetyl-CoA carboxylase (Acc1) within the StH genome species, two Acc1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from 35 diploid taxa representing 19 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) the StH genome species from the same areas or neighboring geographic regions are closely related to each other; (2) the Acc1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) Dasypyrum has contributed to the nuclear genome of Elymus repens and Elymus mutabilis; (4) the StH genome polyploids have higher levels of sequence diversity in the H genome homoeolog than the St genome homoeolog; and (5) the Acc1 sequence may evolve faster in the polyploid species than in the diploids. Our result provides some insight on evolutionary dynamics of duplicate Acc1 gene, the polyploidy speciation and phylogeny of the StH genome species.     

Ribosomal DNA Comparisons of Globodera from Two Continents

Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3'' end of the 18S gene and the 5'' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.     

Divergence of the mitochondrial electron transport chains from the green alga Chlamydomonas reinhardtii and its colorless close relative Polytomella sp.

Compelling evidence exists that the colorless algae of the genus Polytomella arose from a green Chlamydomonas-like ancestor by losing its functional photosynthetic apparatus. Due to the close relationship between the colorless and the green chlorophyte, Polytomella sp. appeared as a useful indicative framework for structural studies of Chlamydomonas reinhardtii mitochondria. However, comparative studies reported here unexpectedly revealed significant differences between the mitochondrial respiratory systems of the two algae. Two-dimensional blue native/SDS-PAGE of isolated mitochondria indicated that cytochrome-containing respiratory complexes III and IV in the two chlorophytes contrast in size, subunit composition and relative abundance. Complex IV in Polytomella is smaller than its counterpart in C. reinhardtii and occurs in two forms that differ presumably in the presence of subunit COXIII. The cytochrome c and the iron-sulfur Rieske protein of both chlorophytes revealed structural differences on the amino acid sequence level. Under comparable culture conditions, the colorless alga exhibits lower levels of cytochrome c and complex IV but a higher respiratory activity than the green alga. Cytochrome c levels were also found to be differently regulated by the growth conditions in both algae. The divergence between the respiratory systems in the two related chlorophytes can be viewed as a consequence of the loss of photosynthetic activity and/or of the adaptation to the environment via the acquisition of a more flexible, heterotrophic metabolism. Our understanding of mitochondrial function and evolution is expected to be greatly enhanced via further parallel studies of photosynthetic/non-photosynthetic algae, for which this study forms an incentive.     

Phylogenetic Relationships of Globodera millefolii,G. artemisiae,and Cactodera salina Based on ITS Region of Ribosomal DNA

Globodera millefolii and G. artemisiae are interesting because their type localities (Estonia and Russia, respectively) are geographically distant from those of the potato cyst nematodes and other Globodera species that seem to have originated in the Western world, and because the type host for each is a member of Compositae rather than Solanaceae. Sequence data for ITS1, ITS2, and 5.8S ribosomal DNA (ITS rDNA) for G. millefolii and G. artemisiae were nearly identical to sequence data for Cactodera salina from the rhizosphere of the estuary plant Salicornia bigelovii in Sonora, Mexico. The ITS rDNA sequences of these three species were all about 94% similar to those of two other Cactodera species for which ITS rDNA data were obtained. Phylogenetic analysis indicated that, based on the ITS rDNA data, G. millefolii and G. artemisiae are more closely related phylogenetically to the Cactodera species than to other nominal Globodera species. The molecular data further suggest that the genus Cactodera may comprise two or more morphologically similar but separate groups.     

Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant

Arabidopsis thaliana APETALA3 (AP3) and Antirrhinum majus DEFICIENS (DEF) MADS box genes are required to specify petal and stamen identity. AP3 and DEF are members of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. In order to investigate the molecular mechanisms underlying organ development in early diverging clades of core eudicots, we isolated and identified an AP3 homolog, FaesAP3, from Fagopyrum esculentum (buckwheat, Polygonaceae), a multi-food-use pseudocereal with healing benefits. Protein sequence alignment and phylogenetic analyses revealed that FaesAP3 grouped into the euAP3 lineage. Expression analysis showed that FaesAP3 was transcribed only in developing stamens, and differed from AP3 and DEF, which expressed in developing petals and stamens. Moreover, ectopic expression of FaesAP3 rescued stamen development without complementation of petal development in an Arabidopsis ap3 mutant. Our results suggest that FaesAP3 is involved in the development of stamens in buckwheat. These results also suggest that FaesAP3 holds some potential for biotechnical engineering to create a male sterile line of F. esculentum.     

Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena

A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2000 μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.     

A pathogenic mutation in cytochrome c oxidase results in impaired proton pumping while retaining O2-reduction activity

In this work we have investigated the effect of a pathogenic mitochondrial DNA mutation found in human colon cells, at a functional-molecular level. The mutation results in the amino-acid substitution Tyr19His in subunit I of the human CytcO and it is associated with respiratory deficiency. It was introduced into Rhodobacter sphaeroides, which carries a cytochrome c oxidase (cytochrome aa₃) that serves as a model of the mitochondrial counterpart. The residue is situated in the middle of a pathway that is used to transfer substrate protons as well as protons that are pumped across the membrane. The Tyr33His (equivalent residue in the bacterial CytcO) structural variant of the enzyme was purified and its function was investigated. The results show that in the structurally altered CytcO the activity decreased due to slowed proton transfer; proton transfer from an internal proton donor, the highly-conserved Glu286, to the catalytic site was slowed by a factor of ∼ 5, while reprotonation of the Glu from solution was slowed by a factor of ∼ 40. In addition, in the structural variant proton pumping was completely impaired. These results are explained in terms of introduction of a barrier for proton transfer through the D pathway and changes in the coordination of water molecules surrounding the Glu286 residue. The study offers an explanation, at the molecular level, to the link between a specific amino-acid substitution and a pathogenic phenotype identified in human colon cells.     

Intraspecific variation of the crotamine and crotasin genes in Crotalus durissus rattlesnakes

Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with β-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5–29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r² = 0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.     

Comparisons of Isolates of Heterodera avenae using 2-D PAGE Protein Patterns and Ribosomal DNA

Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.     

Phylogenetic Relationships Based on Ribosomal DNA Data for Four Species of Cyst Nematodes from Italy and One from Syria

Phylogenetic analysis of new ribosomal DNA (rDNA) data for Heterodera mediterranea, H. hordecalis, H. carotae, and H. fici from Italy and H. ciceri from Syria, along with published data for other species, showed high bootstrap support for the following relationships: (((((H. carotae H. cruciferae) H. goettingiana) (((H. trifolii H. ciceri) H. mediterranea) ((H. avenae H. latipons) H. fici))) (Cactodera betulae H. hordecalis)) (Globodera rostochiensis G. pallida)). The rDNA sequence data were for the two internal transcribed spacers (ITS1 and ITS2) plus the 5.8S gene between them. These inferred relationships support the classic ''''Goettingiana Group'''' of H. carotae, H. cruciferae, and H. goettingiana. A clade comprised of Cactodera betulae and H. hordecalis is only distantly related to the other species in the analysis.     

Phylogenetic Relationships Among Selected Heteroderoidea Based on 18S and ITS Ribosomal DNA

In a study of relationships among selected cyst-forming and noncyst-forming species of Heteroderoidea, combined sequences comprised of DNA from part of the conserved 18S ribosomal RNA gene (rDNA) plus the complete ITS rDNA segment were more similar to analyses based on the ITS data alone than to analyses based on the 18S data alone. One of the two noncyst-forming species, Ekphymatodera thomasoni, grouped with cyst-forming species of Heteroderoidea. Bilobodera flexa, also a noncyst-forming species, was separated from all the other taxa by a long branch. Afenestrata koreana, with a weakly sclerotized cyst, grouped closely with H. bifenestra. These observations suggest that phylogenetic analyses using molecular data may aid in our understanding of the evolution of cyst formation in nematodes, including the possibility of secondary loss. The usefulness of molecular phylogenetic analyses in nematodes may depend more on the particular selection of taxa than on mere addition of data from additional genes.     

Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species diversification and distribution of key morphological traits inferred from dated molecular phylogenetic trees

Background and Aims

Previous work on the pantropical genus Ixora has revealed an Afro-Madagascan clade, but as yet no study has focused in detail on the evolutionary history and morphological trends in this group. Here the evolutionary history of Afro-Madagascan Ixora spp. (a clade of approx. 80 taxa) is investigated and the phylogenetic trees compared with several key morphological traits in taxa occurring in Madagascar.

Methods

Phylogenetic relationships of Afro-Madagascan Ixora are assessed using sequence data from four plastid regions (petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil evidence.

Key Results

Two separate lineages of Madagascan taxa are recovered, one of which is nested in a group of East African taxa. Divergence in Ixora is estimated to have commenced during the mid Miocene, with extensive cladogenesis occurring in the Afro-Madagascan clade during the Pliocene onwards.

Conclusions

Both lineages of Madagascan Ixora exhibit morphological innovations that are rare throughout the rest of the genus, including a trend towards pauciflorous inflorescences and a trend towards extreme corolla tube length, suggesting that the same ecological and selective pressures are acting upon taxa from both Madagascan lineages. Novel ecological opportunities resulting from climate-induced habitat fragmentation and corolla tube length diversification are likely to have facilitated species radiation on Madagascar.     

The complete mitochondrial genome sequence and gene organization of the mud crab (Scylla paramamosain) with phylogenetic consideration

The complete mitochondrial genome is of great importance for better understanding the genome-level characteristics and phylogenetic relationships among related species. In the present study, we determined the complete mitochondrial genome DNA sequence of the mud crab (Scylla paramamosain) by 454 deep sequencing and Sanger sequencing approaches. The complete genome DNA was 15,824 bp in length and contained a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). Of 37 genes, twenty-three were encoded by the heavy strand (H-strand), while the other ones were encoded by light strand (L-strand). The gene order in the mitochondrial genome was largely identical to those obtained in most arthropods, although the relative position of gene tRNA^His differed from other arthropods. Among 13 protein-coding genes, three (ATPase subunit 6 (ATP6), NADH dehydrogenase subunits 1 (ND1) and ND3) started with a rare start codon ATT, whereas, one gene cytochrome c oxidase subunit I (COI) ended with the incomplete stop codon TA. All 22 tRNAs could fold into a typical clover-leaf secondary structure, with the gene sizes ranging from 63 to 73 bp. The phylogenetic analysis based on 12 concatenated protein-coding genes showed that the molecular genetic relationship of 19 species of 11 genera was identical to the traditional taxonomy.     

Hebeloma species associated with Cistus

The genus Hebeloma has a number of species highly specific to Cistus and others that occur with several host genera. This paper discusses the species of Hebeloma that appear to be ectomycorrhizal with Cistus, judging from their occurrence when Cistus is the only available host. The previously unknown species H. plesiocistum spec. nov. is described. We also provide a key to the known Hebeloma associates of Cistus. Molecular analyses based on ITS sequence data further illustrate the distinctness of the newly described species and difficulties in the species delimitation with view to H. erumpens. Specific associations with Cistus may have evolved more than once within the genus Hebeloma.     

The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.