New haplotype of the complete mitochondrial genome of <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> and its species-specific DNA markers: distinguishing <Emphasis Type="Italic">C. siamensis</Emphasis> from <Emphasis Type="Italic">C. porosus</Emphasis> in Thailand

Based on molecular phylogeny of available complete mitochondrial DNA (mtDNA) genome sequences reveals that Crocodylus siamensis and C. porosus are closely related species. Yet, the sequence divergence of their mtDNA showed only a few values under conspecific level. In this study, a new haplotype (haplotype2, EF581859) of the complete mtDNA genome of Siamese crocodile (C. siamensis) was determined. The genome organization, which appeared to be highly similar to haplotype1 (DQ353946) mtDNA genome of C. siamensis, was 16,814 bp in length. However, the sequence divergence between the two genomes differed by around 7–10 and 0.7–2.1% for the haplotype1 between C. siamensis and C. porosus (AJ810453). These results were consistent with the phylogenetic relationship among the three genomes, suggesting that C. siamensis haplotype1 mtDNA genome might be the hybrid or the intraspecific variation of C. porosus. On the other hand, our specimen was found to be a true C. siamensis. Simultaneously, the seven species-specific DNA markers designed based on the distinctive site between haplotype2 mtDNA sequences of C. siamensis and haplotype1 mtDNA sequence of C. siamensis–C. porosus were successfully used to distinguish C. siamensis from C. porosus. These effective markers could be used primarily for rapid and accurate species identification in population, ecology and conservation studies.     

Phylogeny of <Emphasis Type="Italic">Banana Streak Virus</Emphasis> Reveals Recent and Repetitive Endogenization in the Genome of Its Banana Host (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Banana streak virus (BSV) is a plant dsDNA pararetrovirus (family Caulimoviridae, genus badnavirus). Although integration is not an essential step in the BSV replication cycle, the nuclear genome of banana (Musa sp.) contains BSV endogenous pararetrovirus sequences (BSV EPRVs). Some BSV EPRVs are infectious by reconstituting a functional viral genome. Recent studies revealed a large molecular diversity of episomal BSV viruses (i.e., nonintegrated) while others focused on BSV EPRV sequences only. In this study, the evolutionary history of badnavirus integration in banana was inferred from phylogenetic relationships between BSV and BSV EPRVs. The relative evolution rates and selective pressures (d_N/d_S ratio) were also compared between endogenous and episomal viral sequences. At least 27 recent independent integration events occurred after the divergence of three banana species, indicating that viral integration is a recent and frequent phenomenon. Relaxation of selective pressure on badnaviral sequences that experienced neutral evolution after integration in the plant genome was recorded. Additionally, a significant decrease (35%) in the EPRV evolution rate was observed compared to BSV, reflecting the difference in the evolution rate between episomal dsDNA viruses and plant genome. The comparison of our results with the evolution rate of the Musa genome and other reverse-transcribing viruses suggests that EPRVs play an active role in episomal BSV diversity and evolution.     

Mitochondrial genome sequence of <Emphasis Type="Italic">Unionicola parkeri</Emphasis> (Acari: Trombidiformes: Unionicolidae): molecular synapomorphies between closely-related <Emphasis Type="Italic">Unionicola</Emphasis> gill mites

The mitochondrial genome of Unionicola parkeri is a 14,734 bp circular DNA molecule. The sequence and annotation revealed a unique gene order, related to but distinct from the gene order in the closely related species U. foili. Mitochondrial tRNA sequences annotated in this genome predict non-canonical secondary structures for these molecules. The continuing patterns of unique gene orders and unusual tRNA structures in the Trombidiformes in general and Unionicola in particular support the use of phylogenetic approaches that use these types of molecular features as shared, derived character states. Further progress in using these molecular character states to reconstruct phylogeny will depend on careful annotation, especially of tRNA genes.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Mitochondrial Genome of <Emphasis Type="Italic">Savalia savaglia</Emphasis> (Cnidaria,Hexacorallia) and Early Metazoan Phylogeny

Mitochondrial genomes have recently become widely used in animal phylogeny, mainly to infer the relationships between vertebrates and other bilaterians. However, only 11 of 723 complete mitochondrial genomes available in the public databases are of early metazoans, including cnidarians (Anthozoa, mainly Scleractinia) and sponges. Although some cnidarians (Medusozoa) are known to possess atypical linear mitochondrial DNA, the anthozoan mitochondrial genome is circular and its organization is similar to that of other metazoans. Because the phylogenetic relationships among Anthozoa as well as their relation to other early metazoans still need to be clarified, we tested whether sequencing the complete mitochondrial genome of Savalia savaglia, an anthozoan belonging to the order Zoantharia (=Zoanthidea), could be useful to infer such relationships. Compared to other anthozoans, S. savaglia’s genome is unusually long (20,766 bp) due to the presence of several noncoding intergenic regions (3691 bp). The genome contains all 13 protein coding genes commonly found in metazoans, but like other Anthozoa it lacks most of the tRNAs. Phylogenetic analyses of S. savaglia mitochondrial sequences show Zoantharia branching closely to other Hexacorallia, either as a sister group to Actiniaria or as a sister group to Actiniaria and Scleractinia. The close relationships suggested between Zoantharia and Actiniaria are reinforced by strong similarities in their gene order and the presence of similar introns in the COI and ND5 genes. Our study suggests that mitochondrial genomes can be a source of potentially valuable information on the phylogeny of Hexacorallia and may provide new insights into the evolution of early metazoans. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Axel Meyer]     

Genotypic and phenotypic analysis of strains assigned to the widespread cyanobacterial morphospecies <Emphasis Type="Italic">Phormidium </Emphasis><Emphasis Type="Italic">autumnale</Emphasis> (Oscillatoriales)

In this study, ten cyanobacterial strains assigned to the oscillatorian species Phormidium autumnale have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, and pigment content. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The strains were quite homogenous in their morphologic features. Their thylakoids showed a stacked or fascicular pattern. Some, but not all strains contained phycoerythrin. Only one strain (P. autumnale UTCC 476) deviated significantly in its phenotype by lacking a calyptra. In neighbour-joining and maximum Parsimony trees most 16S rRNA sequences were located on a single well-defined branch, which, however, also harboured sequences assigned to other cyanobacterial genera. Two strains (P. autumnale UTCC 476 and P. autumnale UTEX 1580) were found on distant branches. The presence of phycoerythrin was not correlated with the strains’ position in the phylogenetic trees. Our results reconfirm that the morphospecies P. autumnale and the Phormidium group in general are not phylogenetically coherent and require revision. However, as indicated by sequence similarities most of the strains assigned to P. autumnale except P. autumnale UTCC 476 and P. autumnale UTEX 1580 are phylogenetically related and might belong to a single genus.     

Evaluating <Emphasis Type="Italic">Theobroma grandiflorum</Emphasis> for comparative genomic studies with <Emphasis Type="Italic">Theobroma cacao</Emphasis>

The seeds of Theobroma cacao (cacao) are the source of cocoa, the raw material for the multi-billion dollar chocolate industry. Cacao’s two most important traits are its unique seed storage triglyceride (cocoa butter) and the flavor of its fermented beans (chocolate). The genome of T. cacao is being sequenced, and to expand the utility of the genome sequence to the improvement of cacao, we are evaluating Theobroma grandiflorum, the closest economically important species of Theobroma for its potential use in a comparative genomic study. T. grandiflorum differs from cacao in important agronomic traits such as flavor of the fermented beans, disease resistance to witches’ broom and abscission of mature fruits. By comparing genomic sequences and analyzing viable inter-specific hybrids, we hope to identify the key genes that regulate cacao’s most important traits. We have investigated the utility in T. grandiflorum of three types of markers (microsatellite markers, single-strand conformational polymorphism markers and single nucleotide polymorphism (SNP) markers) developed in cacao. Through sequencing of amplicons of 12 diverse individuals of both cacao and T. grandiflorum, we have identified new intra- and inter-specific SNPs. Two markers which had no overlap of alleles between the species were used to genotype putative inter-specific hybrid seedlings. Sequence conservation was significant and species-specific differences numerous enough to suggest that comparative genomics of T. grandiflorum and T. cacao will be useful in elucidating the genetic differences that lead to a variety of important agronomic trait differences.     

Evolution of <Emphasis Type="Italic">mal</Emphasis> ABC transporter operons in the <Emphasis Type="Italic">Thermococcales</Emphasis> and <Emphasis Type="Italic">Thermotogales</Emphasis>

Background  

The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry.     

Rapid evolution of promoters for the plastome gene <Emphasis Type="Italic">ndhF</Emphasis> in flowering plants

Plastome is thought to be a very conservative part of plant genome but little is known about the evolution of plastome promoters. It was previously shown that one light-regulated promoter (LRPpsbD) is highly conserved in different flowering plant species and in black pine. We have undertaken search and demonstrated that gene ndhF is located in a plastome region that rarely underwent substantial rearrangements in terrestrial plants. However, alignment of sequences upstream ndhF suggests that promoters of this gene underwent comparatively rapid evolution in flowering plants. Probably, the ancestor of two basal Magnoliophyta branches (magnoliids and eudicotyledons) had the promoter P_A-ndhF, which was substituted with other promoters—P_B-ndhF and P_C-ndhF—in some phylogenetic lineages of dicots. We failed to reveal conservative sequences with potential promoters of −10/−35 type upstream ndhF genes of monocotyledonous plants, including nine representatives of the grass family (Poaceae). Multiple alignments of sequences from related taxa showed that the predicted ndhF promoters (A–C) underwent frequent mutations and these mutations are not only nucleotide substitutions but also small insertions and deletions. Thus, we can assume that at least some plastome promoters evolve rapidly.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Phylogenetic study of Oryzoideae species and related taxa of the Poaceae based on <Emphasis Type="Italic">atpB</Emphasis>-<Emphasis Type="Italic">rbcL</Emphasis> and <Emphasis Type="Italic">ndhF</Emphasis> DNA sequences

Oryzoideae (Poaceae) plants have economic and ecological value. However, the phylogenetic position of some plants is not clear, such as Hygroryza aristata (Retz.) Nees. and Porteresia coarctata (Roxb.) Tateoka (syn. Oryza coarctata). Comprehensive molecular phylogenetic studies have been carried out on many genera in the Poaceae. The different DNA sequences, including nuclear and chloroplast sequences, had been extensively employed to determine relationships at both higher and lower taxonomic levels in the Poaceae. Chloroplast DNA ndhF gene and atpB-rbcL spacer were used to construct phylogenetic trees and estimate the divergence time of Oryzoideae, Bambusoideae, Panicoideae, Pooideae and so on. Complete sequences of atpB-rbcL and ndhF were generated for 17 species representing six species of the Oryzoideae and related subfamilies. Nicotiana tabacum L. was the outgroup species. The two DNA datasets were analyzed, using Maximum Parsimony and Bayesian analysis methods. The molecular phylogeny revealed that H. aristata (Retz.) Nees was the sister to Chikusichloa aquatica Koidz. Moreover, P. coarctata (Roxb.) Tateoka was in the genus Oryza. Furthermore, the result of evolution analysis, which based on the ndhF marker, indicated that the time of origin of Oryzoideae might be 31 million years ago.     

<Emphasis Type="Italic">Colletotrichum echinochloae</Emphasis>, a new species on Japanese barnyard millet (<Emphasis Type="Italic">Echinochloa utilis</Emphasis>)

Five isolates of a species of Colletotrichum were collected from Japanese barnyard millet (Echinochloa utilis) in Japan. Although the fungus had once been identi-fied as C. graminicola sensu lato, it was clearly different from C. graminicola isolated from maize (Zea mays) in its falcate and short conidia, 18.0–22.2 μm in length, cultural characteristics, and specific pathogenicity to E. utilis. Moreover, molecular phylogenetic analyses using sequences of rDNA-ITS, HMG, and SOD2 indicated a monophyly of the isolates. A new species, Colletotrichum echinochloae, is then proposed based on the morphological, pathological, and molecular characteristics.     

Polymorphism and selection in the major histocompatibility complex <Emphasis Type="Italic">DRA</Emphasis> and <Emphasis Type="Italic">DQA</Emphasis> genes in the family Equidae

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.     

Structure and Evolution of the Atypical Mitochondrial Genome of <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> (Isopoda,Crustacea)

The crustacean isopod Armadillidium vulgare is characterized by an unusual ∼42-kb-long mitochondrial genome consisting of two molecules co-occurring in mitochondria: a circular ∼28-kb dimer formed by two ∼14-kb monomers fused in opposite polarities and a linear ∼14-kb monomer. Here we determined the nucleotide sequence of the fundamental monomeric unit of A. vulgare mitochondrial genome, to gain new insight into its structure and evolution. Our results suggest that the junction zone between monomers of the dimer structure is located in or near the control region. Direct sequencing indicated that the nucleotide sequences of the different monomer units are virtually identical. This suggests that gene conversion and/or replication processes play an important role in shaping nucleotide sequence variation in this mitochondrial genome. The only heteroplasmic site we identified predicts an alloacceptor tRNA change from tRNA^Ala to tRNA^Val. Therefore, in A. vulgare, tRNA^Ala and tRNA^Val are found at the same locus in different monomers, ensuring that both tRNAs are present in mitochondria. The presence of this heteroplasmic site in all sequenced individuals suggests that the polymorphism is selectively maintained, probably because of the necessity of both tRNAs for maintaining proper mitochondrial functions. Thus, our results provide empirical evidence for the tRNA gene recruitment model of tRNA evolution. Moreover, interspecific comparisons showed that the A. vulgare mitochondrial gene order is highly derived compared to the putative ancestral arthropod type. By contrast, an overall high conservation of mitochondrial gene order is observed within crustacean isopods.     

Molecular phylogeny of the blind cavefish <Emphasis Type="Italic">Phreatichthys andruzzii</Emphasis> and <Emphasis Type="Italic">Garra barreimiae</Emphasis> within the family Cyprinidae

The phylogenetic relationships of two cavefish, Phreatichthys andruzzii and Garra barreimiae, belonging to the family Cyprinidae, were investigated by sequencing the mitochondrial cytochrome b gene. These cavefish species are native to Somalia (eastern Africa) and Oman (southeastern Arabian peninsula), respectively, and so far no molecular support to their taxonomy and phylogenetic position was ever provided. The analysis of cytochrome b sequences showed that the species are monophyletic taxa, closely related to each other and to other species of the genus Garra. Molecular clock calculations allowed to date the origin of these hypogaean species back to the Plio-Pleistocene and support the hypothesis that African cyprinids originated from Miocenic immigrations of Asian ancestors.     

Complete sequence and gene organization of the mitochondrial genome of scaly-sided merganser (<Emphasis Type="Italic">Mergus squamatus</Emphasis>) and phylogeny of some Anatidae species

The scaly-sided merganser (Mergus squamatus) is an endangered bird species on the IUCN Red List with the estimated global population of less than 2,500 individuals at present. In the present study, we studied the complete mitochondrial genome (mtDNA) and the phylogenetic of M. squamatus by PCR amplification and GenBank data. The genome was 16,595 bp in length and contained 37 genes (13 protein coding genes, two rRNAs, and 22 tRNAs) and a non-coding control region (D-loop). All protein-coding genes of M. squamatus mtDNA start with a typical ATG codon, except ND1, COI, and COII uses GTG as their initial codon. TAA, T- and TAG as the terminate codon occurred very commonly in the sequence. All tRNA genes can be folded into canonical cloverleaf secondary structure except for tRNA^Ser (AGY) and tRNA^Leu (CUN), which lose ‘‘DHU’’ arm. The genome sequences had been deposited in GenBank under accession number HQ833701. Based on the concatenated nucleotide sequences of mtDNA genes (Cyt b and D-loop), we reconstructed phylogenetic trees and discussed the phylogenetic relationships among ten Anatidae species. The results are different from the present classification, and we support Lophodytes cucullatus and Mergullus albellus to be members of the genus Mergus.     

The complete mitochondrial genome structure of snow leopard <Emphasis Type="Italic">Panthera uncia</Emphasis>

The complete mitochondrial genome (mtDNA) of snow leopard Panthera uncia was obtained by using the polymerase chain reaction (PCR) technique based on the PCR fragments of 30 primers we designed. The entire mtDNA sequence was 16 773 base pairs (bp) in length, and the base composition was: A—5,357 bp (31.9%); C—4,444 bp (26.5%); G—2,428 bp (14.5%); T—4,544 bp (27.1%). The structural characteristics [0] of the P. uncia mitochondrial genome were highly similar to these of Felis catus, Acinonyx jubatus, Neofelis nebulosa and other mammals. However, we found several distinctive features of the mitochondrial genome of Panthera unica. First, the termination codon of COIII was TAA, which differed from those of F. catus, A. jubatus and N. nebulosa. Second, tRNA^{Ser (AGY)}, which lacked the ‘‘DHU’’ arm, could not be folded into the typical cloverleaf-shaped structure. Third, in the control region, a long repetitive sequence in RS-2 (32 bp) region was found with 2 repeats while one short repetitive segment (9 bp) was found with 15 repeats in the RS-3 region. We performed phylogenetic analysis based on a 3 816 bp concatenated sequence of 12S rRNA, 16S rRNA, ND2, ND4, ND5, Cyt b and ATP8 for P. uncia and other related species, the result indicated that P. uncia and P. leo were the sister species, which was different from the previous findings.     

DNA content in nuclei of <Emphasis Type="Italic">Cyclops kolensis</Emphasis> and <Emphasis Type="Italic">Cyclops insignis</Emphasis> (Crustacea,Copepoda)

Chromatin diminution (CD) in two Cyclopoida species, Cyclops kolensis and Cyclops insignis, was studied by static digital Feulgen cytophotometry. DNA content (pg/cell) was evaluated with standard dependences constructed by amounts of DNA in the blood cells of five organisms with known DNA contents of 1.25–14.7 pg. It was found that the C. kolensis diploid genome had about 40 pg DNA before CD and 1.8–2.0 pg DNA after CD. These values are similar both for Moscow and Baikal population of C. kolensis and exceed previous estimates by six to ten times (Grishanin, 2008). Our data confirm that CD reaches 94–96% of DNA content in C. kolensis. In mitotic cells of C. insignis DNA content was about 7.5 pg in both early and late embryos; CD was not revealed for this species. The data obtained show that the DNA content in the C. kolensis genome before CD is highest among the examined Cyclopoides.